Compostions and methods for treating chemosensory dysfunction

ABSTRACT

The invention is directed to a method of detecting a biological substance in the nasal secretion and diagnosing a disease following the detection of the biological substance wherein the biological substance is not related to a respiratory disease. The invention also provides treatment of the diseases following the detection of the biological substance and/or diagnosis of the disease. In some embodiments, the diseases are cancer, hepatitis, smell loss, taste loss, diabetes, and leprosy. The invention also provides a kit for diagnosing a disease. The present invention includes methods of analyzing samples from the nose for the detection of biological substances. In particular, nasal secretion or nasal mucus is collected and analyzed for biological substances. The results of this analysis are then suitable for use in diagnosis, prognosis, and determination of suitability of therapeutic interventions.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 13/932,613, filed Jul. 1, 2013, now U.S. Pat. No. 8,963,706; whichis a continuation application of U.S. application Ser. No. 12/649,320,filed Dec. 29, 2009, now U.S. Pat. No. 8,506,934; which is a divisionalapplication of U.S. application Ser. No. 11/415,942, filed May 1, 2006,now U.S. Pat. No. 7,670,849; which claims priority to U.S. ProvisionalApplication No. 60/743,495, filed Mar. 15, 2006, and U.S. ProvisionalApplication No. 60/676,252, filed Apr. 29, 2005, each of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Detection and identification of biological substances in tissue samplesis used for the diagnosis, prognosis, and monitoring of diseases.Efficient identification of biological substances aids in devisingeffective treatment strategies.

Most of the current diagnostic techniques involve invasive proceduresfor the removal of tissue samples. Hence, there is need for thedevelopment of minimally invasive procedures for biological sampleretrieval.

The present invention provides methods for detection of biologicalsubstances, diagnosis of diseases based on this detection and methodsfor treatment of the diseases after the diagnosis.

SUMMARY OF THE INVENTION

One aspect of the present invention is a method of diagnosing a diseaseby obtaining a nasal specimen, detecting a biological substance in thespecimen, and diagnosing the disease, wherein the diagnosis is based onthe detection of the biological substance, and wherein the biologicalsubstance is not related to a respiratory disease. In some embodiments,the biological substance is a nucleic acid. In some preferredembodiments, the nucleic acid is DNA, RNA or a combination thereof. Insome embodiments, the detection is by a nucleic acid detection method.In some embodiments, the nucleic acid detection method is selected fromthe group consisting of polymerase chain reaction (PCR), ligase chainreaction (LCR), strand displacement amplification (SDA), self-sustainedsequence replication (3SR), array based test, and TAQMAN. In somepreferred embodiments, the nucleic acid detection method is PCR.

In some embodiments of the present invention, the biological substanceis selected from the group consisting of insulin, insulin receptor,leptin, agouti-related protein, ghrelin, glucose, growth factor,caspase, adenylyl cyclase, and carbonic anhydrase. In some embodiments,the biological substance is selected from the group consisting of p53,or mutated p53, TNFα, TNFR I, TNFR II, TRAIL, IL3, endostatin,erythropoietin, bone morphogenic protein, brain derived neurotrophicfactor, ciliary neurotrophic factor, granulocyte macrophage growthfactor, hepatocyte growth factor, platelet derived growth factor,carbonic anhydrase VI, cAMP, cGMP, nitric oxide, insulin like growthfactor, and endoglin. In some preferred embodiments, the biologicalsubstance is TNFα. In some preferred embodiments, the biologicalsubstance is insulin or insulin receptor. In some preferred embodiments,the biological substance is leptin or agouti-related protein. In somepreferred embodiments, the biological substance is TRAIL or carbonicanhydrase VI. In some preferred embodiments, the biological substance isp53.

One aspect of the present invention is method of treating a diseasewherein the treatment is based on diagnosing the disease, wherein thediagnosis is based on a detection of a biological substance in a nasalspecimen, and wherein the biological substance is not related to arespiratory disease. In some embodiments, the disease is selected fromthe group consisting of smell loss, taste loss, diabetes, obesity,anorexia, cancer, leprosy, and hepatitis. In some preferred embodiments,the cancer is ovarian cancer. In some embodiments, the treatment isselected from the group consisting of oral administration, transmucosaladministration, buccal administration, nasal administration, parentaladministration, intravenous, subcutaneous, intramuscular, sublingual,transdermal administration, and rectal administration. In some preferredembodiments, the treatment is by nasal administration. Further theefficacy of the treatement can be monitored by analysis of the nasalspecimens.

Another aspect of the present invention is a method of providing aconclusion regarding a disease to a patient, a health care provider or ahealth care manager where the conclusion is based on a diagnosis,wherein the diagnosis is based on a detection of a biological substancein a nasal specimen, and wherein the biological substance is not relatedto a respiratory disease.

One aspect of the present invention is a method of diagnosing diabetesin a patient by obtaining a nasal specimen, detecting insulin, insulinreceptor or a combination thereof in the specimen, and diagnosing thediabetes in the patient, wherein the diagnosis is based on the detectionof insulin, insulin receptor or a combination thereof in the nasalspecimen. Another aspect of the present invention is a method ofmonitoring an efficacy of a diabetes therapy in a patient by obtaining anasal specimen, detecting insulin, insulin receptor or a combinationthereof in the specimen, and determining the efficacy of the diabetestherapy, wherein the determining is based on the detection of theinsulin, insulin receptor or a combination thereof in the nasalspecimen.

One aspect of the present invention is a method of diagnosing cancer ina patient by obtaining a nasal specimen, detecting TNFα in the specimen,and diagnosing the cancer in the patient, wherein the diagnosis is basedon the detection of TNFα in the nasal specimen. Another aspect of thepresent invention is a method of monitoring an efficacy of a cancertherapy by obtaining a nasal specimen, detecting TNFα in the specimen,and determining the efficacy of the cancer therapy, wherein thedetermining is based on the detection of TNFα in the nasal specimen. Yetanother aspect of the present invention is a method of treating cancerby obtaining a nasal specimen, detecting TNFα in the specimen, andadministering a drug to the patient, wherein the drug modulates theTNFα.

One aspect of the present invention is a method of diagnosing smell lossin a subject by obtaining a nasal specimen, detecting a TNFα, TRAIL,adenylyl cyclases, or carbonic anhydrase VI in the specimen, anddiagnosing the smell loss in the patient, wherein the diagnosis is basedon the detection of TNFα, TRAIL, adenylyl cyclases, or carbonicanhydrase VI in the nasal specimen. In some embodiments, the smell lossis hyposmia, dyosmia, or anosmia. In some embodiments, the methodfurther comprises of treating the smell loss by administeringtheophylline by inhalation.

One aspect of the present invention is a method of diagnosing hepatitisin a patient by obtaining a nasal specimen, detecting antibodies againsta hepatitis causing virus in the specimen, and diagnosing the hepatitisin the patient wherein the diagnosis is based on the detection ofantibodies against the hepatitis causing virus in the nasal specimen. Insome embodiments, the hepatitis is hepatitis A, hepatitis B, hepatitisC, hepatitis D, hepatitis E, and hepatitis G.

Another aspect of the present invention is a method of treating adisease by obtaining a nasal specimen, detecting a biological substancein the specimen, and administering a drug to the patient, wherein thedrug modulates the biological substance. In some preferred embodiments,the disease is cancer and the biological substance is TNFα. In somepreferred embodiments, the disease is diabetes and the biologicalsubstance is insulin or insulin receptor. In some preferred embodiments,the disease is obesity or anorexia and the biological substance isleptin, ghrelin, or agouti-related protein. In some preferredembodiments, the disease is smell loss or taste loss and the biologicalsubstance is TNFα, TRAIL, adenylyl cyclases, or carbonic anhydrase VI.

Yet another aspect of the present invention is a computer-readablemedium wherein the medium comprises the result of an analysis of abiological substance, wherein the biological substances is detected froma specimen of nasal secretion and the biological substance is notrelated to a respiratory disease. In some embodiments, acomputer-readable medium further comprises of a diagnosis of a disease.In some embodiments, at least one step in the methods of the presentinvention is implemented with a computer.

Yet another aspect of the present invention is a kit for a diagnosis ofa disease which comprises a sterile nasal swab for collection of nasalsecretions, an elongated storage and transport tube for receiving theswab wherein the tube is glass or plastic and the tube having areplaceable end closure, and containing a sterile nutrient medium forisolation of the nasal secretions, a sterile assay solution for additionto the transport tube, and a detector medium for the detection of abiological substance in the nasal secretion wherein the biologicalsubstance is not related to a respiratory disease.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a flow chart showing the steps of the methods of the presentinvention.

FIG. 2 illustrates a computer for implementing selected operationsassociated with the methods of the present invention.

FIG. 3 depicts a kit for a detection of biological substance in a nasalspecimen.

FIG. 4 depicts a polyacrylamide gel electrophoresis of samples as shownin Table 1.

FIG. 5 depict LightCycler melting peak report on results of PCR analysisof two samples of nasal mucus. Fluorescence is plotted on ordinate,temperature on abscissa.

FIG. 6 depicts LightCycler data analysis report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, cycle number on abscissa.

FIG. 7 depicts LightCycler melting peak report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, temperature on abscissa.

FIG. 8 depicts LightCycler data analysis report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is platted onordinate, cycle number on abscissa.

FIG. 9 depicts LightCycler data analysis report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, cycle number on abscissa.

FIG. 10 depicts mass spectroscopic analysis of parotid saliva ofpatients before and after treatment with rTCMS.

FIGS. 11 and 12 reflect a feedback mechanism with effects acting fromnose to brain and from brain to nose.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “diagnosis” as used herein and its grammatical equivalents,means the testing of subjects to determine if they have a particulartrait for use in a clinical decision. Diagnosis includes testing ofsubjects at risk of developing a particular disease resulting frominfection by an infectious organism or a non infectious disease, such ascancer or a metabolic disease. Diagnosis also includes testing ofsubjects who have developed particular symptoms to determine the causeof the symptoms. Diagnosis also includes prognosis, monitoring progressof a disease, and monitoring the efficacy of therapeutic regimens. Theresult of a diagnosis can be used to classify patients into groups forperformance of clinical trials for administration of certain therapies.

The term “drug” as used herein, means any compounds of any degree ofcomplexity that perturbs a biological state, whether by known or unknownmechanisms and whether or not they are used therapeutically. Drugs thusinclude: typical small molecules of research or therapeutic interest;naturally-occurring factors, such as endocrine, paracrine, or autocrinefactors or factors interacting with cell receptors of all types;intracellular factors, such as elements of intracellular signalingpathways; factors isolated from other natural sources; pesticides;herbicides; and insecticides.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. The term also refers to syntheticallygenerated nucleic acid.

The term “pathogen” as used herein includes, viral, bacterial, fungal,prion, microbial, or other material that can be detected using theteachings of the present invention. The term “pathogen” as used hereincan be natural or synthetically generated.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds. The term also refers tosynthetically generated polypeptide, peptide or protein.

The term “treating” and its grammatical equivalents as used hereininclude achieving a therapeutic benefit and/or a prophylactic benefit.By therapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

Methods of the Invention

The present invention includes methods of analyzing samples from thenose for the detection of biological substances. In particular, nasalsecretion or nasal mucus is collected and analyzed for biologicalsubstances. The results of this analysis are then suitable for use indiagnosis, prognosis, and determination of suitability of therapeuticinterventions. The techniques of the present invention allow fordetection of biological substances that are typically not considered tobe present in the nasal area, but are known to be present in otherbiological fluids such as blood, serum, plasma, etc. Use of nasalspecimens provides a minimally invasive manner of obtaining biologicalsamples for analysis. Also, the techniques are used to detect substancesthat are not present in other biological fluids such as blood, serum,plasma, etc., but have been now detected in the nasal area.

The term “biological substance” as used herein, and its grammaticalequivalents, includes cells and their extra-cellular and intra-cellularconstituents. For example, biological substances include pathogens,metabolites, DNA, RNA, lipids, proteins, carbohydrates, receptors,enzymes, hormones, growth factors, growth inhibitory factors, cells,organs, tissues, portions of cells, tissues, or organs, subcellularorganelles, chemically reactive molecules like H⁺, superoxides, ATP,citric acid, protein albumin, as well as combinations or aggregaterepresentations of these types of biological variables. In addition,biological substances can include therapeutic agents such asmethotrexate, steroids, non-steroidal anti-inflammatory drugs, solubleTNF-alpha receptor, TNF-alpha antibody, and interleukin-1 receptorantagonists.

A first aspect of the present invention is a method of detecting abiological substance in a nasal specimen, wherein the biologicalsubstance is not related to a respiratory disease caused by a pathogen.Respiratory diseases caused by pathogens include upper respiratory tractviral infection, upper respiratory tract bacterial infection, bacterialsinusitis, and whooping cough. However, in some embodiments, the methodsof the present invention are suitable for detection of substancesrelated to respiratory diseases that are not caused by pathogens, suchas allergic rhinitis, asthma, and chronic obstructive pulmonary disease.It is understood that although some of these diseases are not caused bypathogens, they can be triggered or worsened by pathogens.

A second aspect of the invention is a method of diagnosing a disease byanalyzing a nasal specimen for a biological substance that is notrelated to a respiratory disease. The results of this analysis can thenbe used for diagnosis, prognosis, and determination of suitability oftherapeutic interventions.

The biological substances that can be detected by the methods of thepresent invention include, but are not limited to, insulin, insulinreceptors, leptin, agouti-related protein, ghrelin, glucose, caspases,adenylyl cyclases, carbonic anhydrases, TNF α, TNFR I, TNFR II, TRAIL,IL3, endostatin, erythropoetin, bone morphogenic protein, brain derivedneurotrophic factor, ciliary neurotrophic factor, granulocyte macrophagegrowth factor, hepatocyte growth factor, platelet derived growth factor,carbonic anhydrase VI, cAMP, cGMP, nitric oxide, insulin like growthfactor, and endoglin.

One embodiment of the invention is the detection of substances relatedto glucose metabolism such as insulin and insulin receptors. Thedetection of insulin, insulin receptors, and glucose is used in thediagnosis insulin resistance related conditions, such as diabetes. Useof nasal specimens for the detection of glucose and insulin provides aminimally invasive technique for diagnosis of diabetes and managingdiabetes care, in contrast to the invasive techniques such asvenipucture.

Another embodiment of the invention is the detection of TNFα in nasalspecimens. Elevated levels of TNFα have been shown to play a role indiverse disease processes. TNFα in nasal mucus was found to be about 30times higher than in saliva. The concentration of TNFα in nasalspecimens, thus, can be reflective of underlying disease processes. Thisdetection is used in the diagnosis of various cancers and inflammatorydiseases. Also, TNFα level monitoring in nasal specimens can be used tomonitor the efficacy of cancer and inflammatory disease therapeutics.Further, nasal administration of anti-TNFα drugs provides a means fortreatment of diseases in which TNFα plays a role. Also, levels of TNFαin nasal specimens can be used to study apoptosis.

Yet another embodiment of the invention is the detection of leptin,ghrelin and agouti-related protein in nasal specimens. Also, the methodsinclude the administration of substances that modulate leptin, ghrelinand agouti-related protein for the control of appetite and treatment ofobesity and anorexia. Nasal administration of leptin can inhibitappetite and nasal administration of agouti-related protein canstimulate appetite. Antibodies to leptin, ghrelin and agouti-relatedprotein can be administered intranasally to modulate appetite. Thismodulation includes control and/or stimulation.

In some embodiments, the methods of the present invention includedetecting cAMP and cGMP in nasal specimens. Comparison of themeasurement of cAMP and cGMP in normal subjects with patients with tasteand smell loss indicated that patients with taste and smell loss haddecreased levels of cAMP in their nasal mucus. Hence, cAMP in nasalmucus can be an index of smell loss. The detection of cAMP and cGMP inthe nasal mucus provides a non-invasive method for the detection oftaste and smell loss in a subject.

A third aspect of the invention is a method of treating a diseasewherein the treatment is based on the diagnosis of the disease byanalyzing a nasal specimen for a biological substance that is notrelated to a respiratory disease. Preferably, following the diagnosis, atherapeutic is administered which modulates the biological specimen. Insome embodiments, the treatment includes nasal administration ofbiological substances, such as, by way of example only, leptin, ghrelin,agouti-related protein, TNFα, insulin, or homones. In some embodiments,the treatment includes nasal administration of therapeutic thatmodulates the identified biological substances.

In one embodiment for treating diabetes and/or insulin resistance,following detection of insulin, insulin receptor and/or glucose, a drugthat modulates the insulin, insulin receptor and/or glucose isadministered. Preferably, this administration is via nose. The drug caninclude antidiabetic drugs, including insulin. In one embodiment fortreating cancer, following detection of TNFα, p53 or mutated p53, a drugthat modulates the TNFα, p53 or mutated p53 is administered. Preferably,this administration is via nose. The drug can include anticancer drugs.In one embodiment for treating leprosy, following detection ofantibodies against mycobacterium leprae, an antibiotic drug isadministered. Preferably, this administration is via nose. In oneembodiment for treating hepatitis, including hepatitis A, B, C, D, E andG, following detection of antibodies against hepatitis causing virus, adrug, such as, by way of example only, interferon is administered.Preferably, this administration is via nose. In one embodiment fortreating obesity, following detection of leptin or agouti-relatedprotein, a drug that modulates the leptin or agouti-related protein isadministered. Preferably, this administration is via nose. The drug caninclude anti-agouti-related protein or leptin. In one embodiment fortreating anorexia, following detection of leptin or agouti-relatedprotein, a drug that modulates the leptin or agouti-related protein isadministered. Preferably, this administration is via nose. The drug caninclude anti-leptin or agouti-related protein. In one embodiment fortreating smell loss or taste loss, following detection of TNFα, CA VI,or TRAIL, a drug that modulates the TNFα, CA VI, or TRAIL isadministered. Preferably, this administration is via nose. The drug caninclude theophylline or other drugs known in the art. In one embodimentfor treating flu, following detection of flu causing pathogen orantibodies against flu causing pathogen, a drug that modulates theinfection is administered. Preferably, this administration is via nose.The drug can include antibiotics or other drugs known in the art.

The biological substances in the body interact with the brain via afeedback mechanism. The biological substances present in the nose aresecreted by glands that are controlled by a brain function and via thefeedback mechanism, these biological substances after secretion, in turnaffect the brain function. The nasal administration of the biologicalsubstance after diagnosing the disease by detecting the biologicalsubstance in the nasal secretion may be reflective of this feedbackmechanism.

In some embodiments, the treatment includes transcranial magneticstimulation (TCMS). TCMS or rTCMS (repetitive TCMS) can induce secretionof biological substances in the body, thereby inducing clinical changes.The patients suffering from loss of taste and/or smell (hypogeusiaand/or hypo smia, respectively) when treated with rTCMS, showedimprovement in their sensory acuity and decrease in their sensorydistortions. Some biological substances in these patients, such as, CAI, II and VI, zinc, and copper were found to be significantly higher inblood plasma, erythrocytes and saliva after treatment with TCMS. Theincrease of the biological substances in the body after TCMS indicatesthat TCMS induces biochemical changes in the body and can be used totreat various diseases including clinical abnormalities of sensoryfunction and neurological disorders.

The methods of the present invention disclosed herein include methodsfor detecting, diagnosing, and treating a disease in a subject, byanalyzing one or more biological substances in nasal tissue orsecretion. The steps of the methods of the present invention aredepicted in FIG. 1. Without limiting the scope of the present invention,the steps can be performed independent of each other or one after theother. One or more steps may be skipped in the methods of the presentinvention. A sample of nasal secretion is collected from a subject atstep 101. One or more biological substances in the specimen is detected,measured and/or analyzed at step 102 by detection techniques known inthe art, such as, PCR, mass spectrometry, protein assays etc. By way ofexample only some of the detection techniques are disclosed herein. Adisease is diagnosed at step 103 based on the detection, measurementand/or analysis of the biological substance. A decision regardingtreatment of the disease is made at step 104, the treatment decisionbeing made based on the diagnosis.

The identification of the biological substances may involve one or morecomparisons with reference specimens. The reference specimen may beobtained from the same subject or from a different subject who is eithernot affected with the disease or is a patient. The reference specimencould be obtained from one subject, multiple subjects or besynthetically generated. The identification may also involve thecomparison of the identification data with the databases to identify thebiological substance.

The steps of the methods of the present invention are provided herein.Without limiting the scope of the present invention, other techniquesfor collection of sample, detection of the biological substances anddiagnosis of the disease are known in the art and are within the scopeof the present invention.

Sample Collection

In the sample collection step, specimens from the nasal area arecollected for analysis. In some embodiments of the invention, a sampleof nasal secretions is collected directly from the nose into acollection tube or device. In other embodiments of the invention, asample of nasal secretion is collected on a sample collection device bypassing it into the nostril of a patient. The device may be insertedsequentially into each nostril of the patient and advanced parallel tothe hard palate with slow rotation. The device is then typicallytransferred to a transport tube, such as a glass or plastic test tube.The transport tube may include a suitable volume of a sterile mediumsuch as ethanol or the like.

Suitable sample collection devices are well known to those skilled inthe art. Preferably, a sample collection device can be a swab, a woodenspatula, bibulous materials such as a cotton ball, filter, or gauze pad,an absorbent-tipped applicator, capillary tube, and a pipette.Preferably, a swab can be used as a sample collection device, and thesample processing element comprises a swab holder or a swab processinginsert. The swab holder or swab processing insert can be tapered orangled to allow a single sample processing element to accommodate alltypes of swabs by allowing swabs with different amounts of fiber, orthat are wound to different levels of tightness, to be held securelywithin the holder or insert. Most preferably, the swab holder or swabprocessing insert securely holds the swab to provide stability.

In some instances, samples may be collected from individuals repeatedlyover a longitudinal period of time (e.g., once a day, once a week, oncea month, biannually or annually). Obtaining numerous samples from anindividual over a period of time can be used to verify results fromearlier detections and/or to identify an alteration as a result of, forexample, drug treatment. Samples can be obtained from humans ornon-humans. Preferably, samples are obtained from humans.

Analysis

In the present invention, a specimen of nasal mucus, secretion, ortissue is collected and analyzed using one or more analytical techniquesincluding enzymatic technique, ELISA, fluorometric technique, massspectrography, HPLC, GLC, PCR, and other similar techniques. The presentinvention also includes methods of diagnosing a disease by analyzingnucleic acids in a nasal specimen by nucleic acid detection methods suchas, but are not limited to polymerase chain reaction (PCR), ligase chainreaction (LCR), strand displacement amplification (SDA), self-sustainedsequence replication (3SR), array based tests, and TAQMAN. A number oftemplate dependent processes are available to amplify the targetsequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159.

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is a process for amplifying one ormore desired specific nucleic acid sequences found in a nucleic acid.Because large amounts of a specific sequence may be produced by thisprocess, it is used for improving the efficiency of cloning DNA ormessenger RNA and for amplifying a target sequence to facilitatedetection thereof.

PCR involves a chain reaction for producing, in exponential quantitiesrelative to the number of reaction steps involved, at least one specificnucleic acid sequence given (a) that the ends of the required sequenceare known in sufficient detail that oligonucleotides can be synthesizedwhich will hybridize to them, and (b) that a small amount of thesequence is available to initiate the chain reaction. The product of thechain reaction would be a discrete nucleic acid duplex with terminicorresponding to the ends of the specific primers employed.

Any source of nucleic acid, in purified or non purified form, can beutilized as the starting nucleic acid or acids, provided it contains oris suspected of containing the specific nucleic acid sequence desired.Thus, the process may employ, for example, DNA or RNA, includingmessenger RNA, which DNA or RNA may be single stranded or doublestranded. In addition, a DNA-RNA hybrid which contains one strand ofeach may be utilized. A mixture of any of these nucleic acids may alsobe employed, or the nucleic acid produced from a previous amplificationreaction herein using the same or different primers may be so utilized.The specific nucleic acid sequence to be amplified may be only afraction of a larger molecule or can be present initially as a discretemolecule, so that the specific sequence constitutes the entire nucleicacid. It is not necessary that the sequence to be amplified be presentinitially in a pure form; it may be a minor fraction of a complexmixture, such as a portion of the β-globin gene contained in whole humanDNA or a portion of nucleic acid sequence due to a particularmicroorganism which organism might constitute only a very minor fractionof a particular biological sample. The starting nucleic acid may containmore than one desired specific nucleic acid sequence which may be thesame or different. Therefore, it is useful not only for producing largeamounts of one specific nucleic acid sequence, but also for amplifyingsimultaneously more than one different specific nucleic acid sequencelocated on the same or different nucleic acid molecules.

The nucleic acid or acids may be obtained from any source, for example,from plasmids such as pBR322, from cloned DNA or RNA, or from naturalDNA or RNA from any source, including bacteria, yeast, viruses, andhigher organisms such as plants or animals. DNA or RNA may be extractedfrom blood, tissue material such as chorionic villi or amniotic cells.

It will be understood that the word primer as used hereinafter may referto more than one primer, particularly in the case where there is someambiguity in the information regarding the terminal sequence(s) of thefragment to be amplified. For instance, in the case where a nucleic acidsequence is inferred from protein sequence information a collection ofprimers containing sequences representing all possible codon variationsbased on degeneracy of the genetic code will be used for each strand.One primer from this collection will be 100% homologous with the end ofthe desired sequence to be amplified.

An appropriate agent may be added for inducing or catalyzing the primerextension reaction and the reaction is allowed to occur under conditionsknown in the art. The inducing agent may be any compound or system whichwill function to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose may include, forexample, E. coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,reverse transcriptase, and other enzymes, including heat-stable enzymes,which will facilitate combination of the nucleotides in the propermanner to form the primer extension products which are complementary toeach nucleic acid strand. Generally, the synthesis can be initiated atthe 3′ end of each primer and proceed in the 5′ direction along thetemplate strand, until synthesis terminates, producing molecules ofdifferent lengths. There may be inducing agents, however, which initiatesynthesis at the 5′ end and proceed in the other direction, using thesame process as described above.

The newly synthesized strand and its complementary nucleic acid strandform a double-stranded molecule which can be used in the succeedingsteps of the process. In the next step, the strands of thedouble-stranded molecule may be separated to provide single-strandedmolecules. New nucleic acid may be synthesized on the single-strandedmolecules. Additional inducing agent, nucleotides and primers may beadded if necessary for the reaction to proceed under the conditionsprescribed above. Again, the synthesis would be initiated at one end ofthe oligonucleotide primers and would proceed along the single strandsof the template to produce additional nucleic acid. After this step,half of the extension product would consist of the specific nucleic acidsequence bounded by the two primers. The steps of strand separation andextension product synthesis can be repeated as often as needed toproduce the desired quantity of the specific nucleic acid sequence. Theamount of the specific nucleic acid sequence produced would accumulatein an exponential fashion. After the appropriate length of time haspassed to produce the desired amount of the specific nucleic acidsequence, the reaction may be halted by inactivating the enzymes in anyknown manner or separating the components of the reaction.

Amplification is useful when the amount of nucleic acid available foranalysis is very small, as, for example, in the prenatal diagnosis ofsickle cell anemia using DNA obtained from fetal cells. Amplification isparticularly useful if such an analysis is to be done on a small sampleusing non-radioactive detection techniques which may be inherentlyinsensitive, or where radioactive techniques are being employed butwhere rapid detection is desirable.

Any known techniques for nucleic acid (e.g., DNA and RNA) amplificationcan be used with the assays described herein. Preferred amplificationtechniques are the polymerase chain reaction (PCR) methodologies whichcomprise solution PCR and in situ PCR.

The invention is not limited to the use of straightforward PCR. A systemof nested primers may be used for example. Other suitable amplificationmethods known in the field can also be applied such as, but not limitedto, ligase chain reaction (LCR), strand displacement amplification(SDA), self-sustained sequence replication (3SR), array based test, andTAQMAN.

As used herein “amplification” may refer to any in vitro method forincreasing the number of copies of a nucleic acid sequence with the useof a DNA polymerase. Nucleic acid amplification results in theincorporation of nucleotides into a DNA molecule or primer therebyforming a new DNA molecule complementary to a DNA template. The newlyformed DNA molecule and its template can be used as templates tosynthesize additional DNA molecules. As used herein, one amplificationreaction may consist of many rounds of DNA replication. DNAamplification reactions include, for example, polymerase chain reactions(PCR). One PCR reaction may consist of 5-100 “cycles” of denaturation,annealing, and synthesis of a DNA molecule.

Fluorescence Microscopy

Some embodiments of the invention include fluorescence microscopy for adetection of a biological substance in a nasal specimen. Fluorescencemicroscopy enables the molecular composition of the structures beingobserved to be identified through the use of fluorescently-labeledprobes of high chemical specificity such as antibodies. It can be doneby directly conjugating a fluorophore to a protein and introducing thisback into a cell. Fluorescent analogue may behave like the nativeprotein and can therefore serve to reveal the distribution and behaviorof this protein in the cell. Along with NMR, infrared spectroscopy,circular dichroism and other techniques, protein intrinsic fluorescencedecay and its associated observation of fluorescence anisotropy,collisional quenching and resonance energy transfer are techniques forprotein detection.

The naturally fluorescent proteins can be used as fluorescent probes.The jellyfish aequorea victoria produces a naturally fluorescent proteinknown as green fluorescent protein (GFP). The fusion of thesefluorescent probes to a target protein enables visualization byfluorescence microscopy and quantification by flow cytometry. Withoutlimiting the scope of the present invention, some of the probes are asfollowing:

Labels: Sensitivity and safety (compared to radioactive methods) offluorescence has led to an increasing use for specific labeling ofnucleic acids, proteins and other biomolecules. Besides fluorescein,other fluorescent labels cover the whole range from 400 to 820 nm. Byway of example only, some of the labels are, fluorescein and itsderivatives, carboxyfluoresceins, rhodamines and their derivatives, attolabels, fluorescent red and fluorescent orange: Cy3/Cy5 alternatives,lanthanide complexes with long lifetimes, long wavelength labels—up to800 nm, DY cyanine labels, and phycobili proteins.

Conjugates: Antibody conjugates can be generated with specificity forvirtually any epitope and are therefore, applicable to imaging a widerange of biomolecules. By way of example only, some of the conjugatesare, isothiocyanate conjugates, streptavidin conjugates, and biotinconjugates.

Enzyme Substrates: By way of example only, some of the enzyme substratesare fluorogenic and chromogenic substrates.

Micro- and Nanoparticles: By way of example only, some of thefluorochromes are: FITC (green fluorescence, excitation/emission=506/529nm), rhodamine B (orange fluorescence, excitation/emission=560/584 nm),and nile blue A (red fluorescence, excitation/emission=636/686 nm).Fluorescent nanoparticles can be used for various types of immunoassays.Fluorescent nanoparticles are based on different materials, such as,polyacrylonitrile, and polystyrene etc.

Molecular Rotors: Fluorescent molecular rotors are sensors ofmicroenvironmental restriction that become fluorescent when theirrotation is constrained. Few examples of molecular constraint includeincreased dye (aggregation), binding to antibodies, or being trapped inthe polymerization of actin.

IEF-Markers: IEF (isoelectric focusing) is an analytical tool for theseparation of ampholytes, mainly proteins. An advantage for IEF-Gelelectrophoresis with fluorescent IEF-marker is the possibility todirectly observe the formation of gradient. Fluorescent IEF-marker canalso be detected by UV-absorption at 280 nm (20° C.).

Any or all of these fluorescent probes can be used for the detection ofbiological substances in the nasal mucus. A peptide library can besynthesized on solid supports and, by using coloring receptors,subsequent dyed solid supports can be selected one by one. If receptorscannot indicate any color, their binding antibodies can be dyed. Themethod can not only be used on protein receptors, but also on screeningbinding ligands of synthesized artificial receptors and screening newmetal binding ligands as well. Automated methods for HTS and FACS(fluorescence activated cell sorter) can also be used. A FACS machineoriginally runs cells through a capillary tube and separate cells bydetecting their fluorescent intensities.

Immunoassays

Some embodiments of the invention include immunoassay for a detection ofa biological substance in a nasal specimen. In immunoblotting like thewestern blot of electrophoretically separated proteins a single proteincan be identified by its antibody. Immunoassay can be competitivebinding immunoassay where analyte competes with a labeled antigen for alimited pool of antibody molecules (e.g. radioimmunoassay, EMIT).Immunoassay can be non-competitive where antibody is present in excessand is labeled. As analyte antigen complex is increased, the amount oflabeled antibody-antigen complex may also increase (e.g. ELISA).Antibodies can be polyclonal if produced by antigen injection into anexperimental animal, or monoclonal if produced by cell fusion and cellculture techniques. In immunoassay, the antibody may serve as a specificreagent for the analyte antigen.

Without limiting the scope and content of the present invention, some ofthe types of immunoassays are, by way of example only, RIAs(radioimmunoassay), enzyme immunoassays like ELISA (enzyme-linkedimmunosorbent assay), EMIT (enzyme multiplied immunoassay technique),microparticle enzyme immunoassay (MEIA), LIA (luminescent immunoassay),and FIA (fluorescent immunoassay). These techniques can be used todetect biological substances in the nasal specimen. Theantibodies—either used as primary or secondary ones—can be labeled withradioisotopes (e.g. 125I), fluorescent dyes (e.g. FITC) or enzymes (e.g.HRP or AP) which may catalyse fluorogenic or luminogenic reactions.

EMIT (Enzyme Multiplied Immunoassay Technique): EMIT is a competitivebinding immunoassay that avoids the usual separation step. A type ofimmunoassay in which the protein is labeled with an enzyme, and theenzyme-protein-antibody complex is enzymatically inactive, allowingquantitation of unlabelled protein.

ELISA (Enzyme Linked Immunosorbent Assay): Some embodiments of theinvention include ELISA to detect biological substances in the nasalspecimen. ELISA is based on selective antibodies attached to solidsupports combined with enzyme reactions to produce systems capable ofdetecting low levels of proteins. It is also known as enzyme immunoassayor EIA. The protein is detected by antibodies that have been madeagainst it, that is, for which it is the antigen. Monoclonal antibodiesare often used.

The test may require the antibodies to be fixed to a solid surface, suchas the inner surface of a test tube, and a preparation of the sameantibodies coupled to an enzyme. The enzyme may be one (e.g.,β-galactosidase) that produces a colored product from a colorlesssubstrate. The test, for example, may be performed by filling the tubewith the antigen solution (e.g., protein) to be assayed. Any antigenmolecules present may bind to the immobilized antibody molecules. Theantibody-enzyme conjugate may be added to the reaction mixture. Theantibody part of the conjugate binds to any antigen molecules that werebound previously, creating an antibody-antigen-antibody “sandwich”.After washing away any unbound conjugate, the substrate solution may beadded. After a set interval, the reaction is stopped (e.g., by adding 1N NaOH) and the concentration of colored product formed is measured in aspectrophotometer. The intensity of color is proportional to theconcentration of bound antigen.

ELISA can also be adapted to measure the concentration of antibodies, inwhich case, the wells are coated with the appropriate antigen. Thesolution (e.g., serum) containing antibody may be added. After it hashad time to bind to the immobilized antigen, an enzyme-conjugatedanti-immunoglobulin may be added, consisting of an antibody against theantibodies being tested for. After washing away unreacted reagent, thesubstrate may be added. The intensity of the color produced isproportional to the amount of enzyme-labeled antibodies bound (and thusto the concentration of the antibodies being assayed).

Radioimmunoassay: Some embodiments of the invention includeradioimmunoassays to detect biological substances in the nasal specimen.Radioactive isotopes can be used to study in vivo metabolism,distribution, and binding of small amount of compounds. Radioactiveisotopes of ¹H, ¹²C, ³¹P, ³²S, and ¹²⁷I in body are used such as ³H,¹⁴C, ³²P, ³⁵S, and ¹²⁵I.

In receptor fixation method in 96 well plates, receptors may be fixed ineach well by using antibody or chemical methods and radioactive labeledligands may be added to each well to induce binding. Unbound ligands maybe washed out and then the standard can be determined by quantitativeanalysis of radioactivity of bound ligands or that of washed-outligands. Then, addition of screening target compounds may inducecompetitive binding reaction with receptors. If the compounds showhigher affinity to receptors than standard radioactive ligands, most ofradioactive ligands would not bind to receptors and may be left insolution. Therefore, by analyzing quantity of bound radioactive ligands(or washed-out ligands), testing compounds' affinity to receptors can beindicated.

The filter membrane method may be needed when receptors cannot be fixedto 96 well plates or when ligand binding needs to be done in solutionphase. In other words, after ligand-receptor binding reaction insolution, if the reaction solution is filtered through nitrocellulosefilter paper, small molecules including ligands may go through it andonly protein receptors may be left on the paper. Only ligands thatstrongly bound to receptors may stay on the filter paper and therelative affinity of added compounds can be identified by quantitativeanalysis of the standard radioactive ligands.

Fluorescence Immunoassays: Some embodiments of the invention includefluorescence immunoassays for a detection of a biological substance in anasal specimen. Fluorescence based immunological methods are based uponthe competitive binding of labeled ligands versus unlabeled ones onhighly specific receptor sites.

The fluorescence technique can be used for immunoassays based on changesin fluorescence lifetime with changing analyte concentration. Thistechnique may work with short lifetime dyes like fluoresceinisothiocyanate (FITC) (the donor) whose fluorescence may be quenched byenergy transfer to eosin (the acceptor). A number of photoluminescentcompounds may be used, such as cyanines, oxazines, thiazines,porphyrins, phthalocyanines, fluorescent infrared-emitting polynucleararomatic hydrocarbons, phycobiliproteins, squaraines and organo-metalliccomplexes, hydrocarbons and azo dyes.

Fluorescence based immunological methods can be, for example,heterogenous or homogenous. Heterogenous immunoassays comprise physicalseparation of bound from free labeled analyte. The analyte or antibodymay be attached to a solid surface. The technique can be competitive(for a higher selectivity) or noncompetitive (for a higher sensitivity).Detection can be direct (only one type of antibody used) or indirect (asecond type of antibody is used). Homogenous immunoassays comprise nophysical separation. Double-antibody fluorophore-labeled antigenparticipates in an equilibrium reaction with antibodies directed againstboth the antigen and the fluorophore. Labeled and unlabeled antigen maycompete for a limited number of anti-antigen antibodies.

Some of the fluorescence immunoassay methods include simple fluorescencelabeling method, fluorescence resonance energy transfer (FRET), timeresolved fluorescence (TRF), and scanning probe microscopy (SPM). Thesimple fluorescence labeling method method can be used forreceptor-ligand binding, enzymatic activity by using pertinentfluorescence, and as a fluorescent indicator of various in vivophysiological changes such as pH, ion concentration, and electricpressure. TRF is a method that selectively measures fluorescence of thelanthanide series after the emission of other fluorescent molecules isfinished. TRF can be used with FRET and the lanthanide series can becomedonors or acceptors. In scanning probe microscopy, in the capture phase,for example, at least one monoclonal antibody is adhered to a solidphase and a scanning probe microscope is utilized to detectantigen/antibody complexes which may be present on the surface of thesolid phase. The use of scanning tunneling microscopy eliminates theneed for labels which normally is utilized in many immunoassay systemsto detect antigen/antibody complexes.

Nuclear Magnetic Resonanace (NMR)

Some embodiments of the invention include NMR for detection of abiological substance in a nasal specimen. NMR spectroscopy is capable ofdetermining the structures of biological macromolecules like proteinsand nucleic acids at atomic resolution. In addition, it is possible tostudy time dependent phenomena with NMR, such as intramolecular dynamicsin macromolecules, reaction kinetics, molecular recognition or proteinfolding. Heteronuclei like ¹⁵N, ¹³C and ²H, can be incorporated inproteins by uniform or selective isotopic labeling. Additionally, somenew information about structure and dynamics of macromolecules can bedetermined with these methods.

X-Ray Crystallography

Some embodiments of the invention include X-ray crystallography fordetection of a biological substance in a nasal specimen. X-raycrystallography is a technique in which the pattern produced by thediffraction of X-rays through the closely spaced lattice of atoms in acrystal is recorded and then analyzed to reveal the nature of thatlattice. This generally leads to an understanding of the material andmolecular structure of a substance. The spacings in the crystal latticecan be determined using Bragg's law. X-ray diffraction is commonlycarried out using single crystals of a material, but if these are notavailable, microcrystalline powdered samples may also be used which mayrequire different equipment.

Fluorescence Spectroscopy

Some embodiments of the invention include fluorescence spectroscopy fordetection of a biological substance in a nasal specimen. By way ofexample only, conventional fluorometry is measurement of emission lightintensities at defined wavelengths for a certain emission maxima of afluorophore. Total fluorometry is a collection of data for a continuumof absorption as well as emission wavelengths. Fluorescence polarizationis when polarized light is used for excitation and binding offluorochrome-labeled antigens to specific antibodies. Line narrowingspectroscopy is low-temperature solid-state spectroscopy that derivesits selectivity from the narrow-line emission spectra.

Time-dependent fluorescence spectroscopy comprises time-resolvedmeasurements containing more information than steady-state measurements,since the steady-state values represent the time average oftime-resolved determinations. It is a single photon timing techniquewhere the time between an excitation light pulse and the first photonemitted by the sample is measured.

Matrix Assisted Laser Desorption Ionization Time-of-Flight MassSpectrometry (MALDI TOF-MS)

Some embodiments of the invention include MALDI TOF-MS for detection ofa biological substance in a nasal specimen. MALDI TOF-MS providesaccurate mass determinations and primary sequence information. Improvedmass resolution in MALDI TOF-MS can be obtained by the utilization of asingle-stage or a dual-stage reflectron (RETOF-MS). In the reflectronmass spectrum, the isotopic multiplet is well resolved producing a fullwidth half maximum (FWHM) mass resolution of about 3400. Massresolutions up to 6000 (FWHM) can be obtained for peptides up to about3000 Da with RETOF-MS. Enhancing the mass resolution can also increasethe mass accuracy when determining the ion's mass.

Both linear and reflectron MALDI-TOF-MS can be utilized for molecularweight determinations of molecular ions and enzymatic digests leading tostructural information of proteins. These digests are typically massanalyzed with or without purification prior to molecular weightdeterminations. Varieties of methodologies have been developed to obtainprimary sequence information for proteins and peptides utilizing MALDITOF-MS. Two different approaches can be taken. The first method is knownas protein ladder sequencing and can be employed to produce structurallyinformative fragments of the analyte prior to insertion into the TOFmass spectrometer and subsequent analysis. The second approach utilizesthe phenomenon of metastable ion decay that occurs inside the TOF massspectrometer to produce sequence information.

The ladder sequencing with TOF-MS consists of either a time-dependent orconcentration-dependent chemical degradation from either the N- orC-terminus of the protein/peptide into fragments, each of which differsby one amino acid residue. The mixture is mass analyzed in a singleMALDI-TOF-MS experiment with mass differences between adjacent massspectral peaks corresponding to a specific amino acid residue. The orderof occurrence in the mass spectrum defines the sequence of amino acidsin the original protein/peptide.

Post-source decay with RETOF-MS MALDI is an ionization technique thatproduces intact protonated pseudomolecular ion species. A significantdegree of metastable ion decay occurs after ion acceleration and priorto detection. The ion fragments produced from the metastable ion decayof peptides and proteins typically include both neutral molecule losses(such as water, ammonia and portions of the amino acid side chains) andrandom cleavage at peptide bonds. In-source decay with linear TOF-MS isan alternative approach to RETOF-MS for studying metastable ion decay ofMALDI generated ions. Primary structural information for peptides andproteins can be obtained by this method. Coherent mass spectral peakscan be produced from these metastable decayed ions giving rise tosignificant structural information for peptides and proteins.

Surface-Enhanced Laser Desorption Ionization—Time of Flight (SELDI-TOF)

Some embodiments of the invention include SELDI TOF-MS for detection ofa biological substance in a nasal specimen. This technique utilizesstainless steel or aluminum-based supports, or chips, engineered withchemical (hydrophilic, hydrophobic, pre-activated, normal-phase,immobilized metal affinity, and cationic or anionic) or biological(antibody, antigen binding fragments (e.g. scFv), DNA, enzyme, orreceptor) bait surfaces of 1-2 mm in diameter. These varied chemical andbiochemical surfaces allow differential capture of proteins based on theintrinsic properties of the proteins themselves. Solubilized tissue orbody fluids in volumes as small as 0.1 μl can be directly applied tothese surfaces, where proteins with affinities to the bait surface maybind. Following a series of washes to remove non-specifically or weaklybound proteins, the bound proteins are laser desorbed and ionized for MSanalysis. Masses of proteins ranging from small peptides of less than1000 Da up to proteins of greater than 300 kDa can be calculated basedon time-of-flight. As mixtures of proteins may be analyzed withindifferent samples, a unique sample fingerprint or signature may resultfor each sample tested. Consequently, patterns of masses rather thanactual protein identifications can be produced by SELDI analysis. Thesemass spectral patterns can be used to differentiate patient samples fromone another, such as diseased from normal.

UV-Vis

Some embodiments of the invention include optical absorptionspectroscopy (UV/VIS) for detection of a biological substance in a nasalspecimen. UV/VIS provides light absorption data which helps in thedetermination of concentration of macromolecules such as, proteins, DNA,nucleotides etc. Organic dyes can be used to enhance the absorption andto shift the absorption into the visible range (e.g. coomassie bluereagents). Resonance raman spectroscopy (RRS) can be used to studymolecular structure and dynamics. RRS helps in investigating specificparts of macromolecules by using different excitation wavelengths.

Liquid Chromatography (LC)

Some embodiments of the invention include LC for a detection ofbiological substance in a nasal specimen. Examples of LC are but notlimited to, affinity chromatography, gel filtration chromatography,anion exchange chromatography, cation exchange chromatography, diodearray-LC and high performance liquid chromatography (HPLC).

Gel filtration chromatography separates proteins, peptides, andoligonucleotides on the basis of size. Molecules may move through a bedof porous beads, diffusing into the beads to greater or lesser degrees.Smaller molecules may diffuse further into the pores of the beads andtherefore move through the bed more slowly, while larger molecules mayenter less or not at all and thus move through the bed more quickly.Both molecular weight and three dimensional shapes contribute to thedegree of retention. Gel Filtration Chromatography may be used foranalysis of molecular size, for separations of components in a mixture,or for salt removal or buffer exchange from a preparation ofmacromolecules.

Affinity chromatography is the process of bioselective adsorption andsubsequent recovery of a compound from an immobilized ligand. Thisprocess allows for the specific and efficient purification of manydiverse proteins and other compounds. Ion exchange chromatographyseparates molecules based on differences between the overall charges ofthe proteins. It can be used for the purification of protein,oligonucleotides, peptides, or other charged molecules.

HPLC can be used in the separation, purification and detection ofbiological substances in the nasal mucus. Crude tissue extracts may beloaded directly onto the HPLC system and mobilized by gradient elution.Rechromatography under the identical conditions is an option if furtherpurification is warranted or necessary. Reversed phase chromatography(RPC) can be utilized in the process of protein structure determination.HPLC may be coupled with MS. The HPLC method described in Henkin et al.,New Frontiers in Immunobiology, 2000, pp. 127-152, is incorporatedherein in its entirety.

The size-exclusion chromatography (SEC) and ion-exchange chromatography(IEC) can be used for separation and purification of biologically activeproteins, such as enzymes, hormones, and antibodies. In liquid affinitychromatography (LAC), interaction may be based on binding of the proteindue to mimicry of substrate, receptor, etc. The protein may be eluted byintroducing a competitive binding agent or altering the proteinconfiguration which may facilitate dissociation. A procedure that can beused in the separation of membrane proteins is the use of nonionicdetergents, such as Triton X-100, or protein solubilization by organicsolvents with IEC.

Diode array detector-liquid chromatography (DAD-LC) provides complete,multiple spectra for each HPLC peak, which, by comparison, can provideindication of peak purity. These data can also assign presence of tyr,trp, phe, and possibly others (his, met, cys) and can quantitate theseamino acids by 2nd derivative or multi-component analysis. By apost-column derivatization, DAD-LC can also identify and quantitate cys,his and arg in individual peptides. Thus, it is possible to analyze for6 of the 20 amino acids of each separated peptide in a single LC run,and information can be obtained about presence or absence of these aminoacids in a given peptide in a single step. This is assisted by knowingthe number of residues in each peptide.

Electrophoresis

Some embodiments of the invention include electrophoresis for detectionof a biological substance in a nasal specimen. Electrophoresis can begel electrophoresis or capillary electrophoresis.

Gel Electrophoresis: Gel electrophoresis is a technique that can be usedfor the separation of proteins. During electrophoresis, macromoleculesare forced to move through pores when an electrical current is applied.Their rate of migration through the electric field depends on strengthof the field, size and shape of the molecules, relative hydrophobicityof the samples, and on an ionic strength and temperature of a buffer inwhich the molecules are moving. After staining, the separatedmacromolecules in each lane can be seen in a series of bands spread fromone end of the gel to the other. Using this technology it is possible toseparate and identify protein molecules that differ by as little as asingle amino acid. Also, gel electrophoresis allows determination ofcrucial properties of a protein such as its isoelectric point andapproximate molecular weight. Electrofocusing or isoelectric focusing isa technique for separating different molecules by their electric chargedifferences (if they have any charge). It is a type of zoneelectrophoresis that takes advantage of the fact that a molecule'scharge changes as the pH of its surroundings changes.

Capillary Electrophoresis: Capillary electrophoresis is a collection ofa range of separation techniques which may involve the application ofhigh voltages across buffer filled capillaries to achieve separations.The variations include separation based on size and charge differencesbetween analytes (termed capillary zone electrophoresis (CZE) or freesolution CE (FSCE)), separation of neutral compounds using surfactantmicelles (micellar electrokinetic capillary chromatography (MECC) orsometimes referred to as MEKC) sieving of solutes through a gel network(capillary gel electrophoresis, GCE), separation of cations (or anions)based on electrophoretic mobility (capillary isotachophoresis, CITP),and separation of zwitterionic solutes within a pH gradient (capillaryisoelectric focusing, CIEF). Capillary electrochromatography (CEC) canbe an associated electrokinetic separation technique which involvesapplying voltages across capillaries filled with silica gel stationaryphases. Separation selectivity in CEC can be a combination of bothelectrophoretic and chromatographic processes. Many of the CE separationtechniques rely on the presence of an electrically induced flow ofsolution (electroosmotic flow, EOF) within the capillary to pump solutestowards the detector.

Arrays

Some embodiments of the invention include arrays for detection of abiological substance in a nasal specimen. Arrays involve performingparallel analysis of multiple samples against known protein targets. Thedevelopment of various microarray platforms can enable and acceleratethe determination of protein abundance, localization, and interactionsin a cell or tissue. Microarrays provide a platform that allowsidentification of protein interaction or function against acharacterized set of proteins, antibodies, or peptides. Protein-basedchips array proteins on a small surface and can directly measure thelevels of proteins in tissues using fluorescence-based imaging. Proteinscan be arrayed on either flat solid phases or in capillary systems(microfluidic arrays), and several different proteins can be applied tothese arrays. In addition to the use of antibodies as array probes,single-stranded oligonucleotides, whose specificity is optimized by invitro elution (aptamers), offer a viable alternative. Nonspecificprotein stains can be then used to detect bound proteins.

Arrays include, but are not limited to, bead arrays, bead based arrays,bioarrays, bioelectronic arrays, cDNA arrays, cell arrays, DNA arrays,gene arrays, gene expression arrays, frozen cell arrays, genome arrays,high density oligonucleotide arrays, hybridization arrays,microcantilever arrays, microelectronic arrays, multiplex DNAhybridization arrays, nanoarrays, oligonucleotide arrays,oligosaccharide arrays, planar arrays, protein arrays, solution arrays,spotted arrays, tissue arrays, exon arrays, filter arrays, macroarrays,small molecule microarrays, suspension arrays, theme arrays, tilingarrays, and transcript arrays.

Sensors

Some embodiments of the invention include sensors for detection of abiological substance in a nasal specimen. Sensors can be used for bothin vivo and in vitro detection. Sensors can be chemical sensors, opticalsensors, and biosensors. Chemical sensors are miniaturized analyticaldevices which may deliver real-time and online information on thepresence of specific compounds or ions in complex samples. Opticalsensors are based on measurement of either intrinsic optical propertiesof analytes, or of optical properties of indicator dyes or labeledbiomolecules attached to solid supports. Biosensors can be affinitybiosensor based on capabilities of enzymes to convert substrates intoproducts or catalytic biosensors. Biosensors detect antibody and analytecomplexes using a variety of physical methods. Some biosensors measurethe change in surface charge that occurs when analyte is bound toantibodies or other binding agents, which in turn are bound to asurface. Other biosensors use binding agents attached to a surface andmeasure a change in a physical property of the support, other thansurface charge, upon binding of analyte. Some biosensor techniques use aspecific property of a labeled binding agent or antigen to produce ameasurable change.

Methods for Identifying Proteins from a Library Screen

Protein identification methods by way of example only includelow-throughput sequencing through Edman degradation, mass spectrometrytechniques, peptide mass fingerprinting, de novo sequencing, andantibody-based assays. The protein quantification assays includefluorescent dye gel staining, tagging or chemical modification methods(i.e. isotope-coded affinity tags (ICATS), combined fractional diagonalchromatography (COFRADIC)). The purified protein may also be used fordetermination of three-dimensional crystal structure, which can be usedfor modeling intermolecular interactions. Common methods for determiningthree-dimensional crystal structure include x-ray crystallography andNMR spectroscopy. Detailed below are a few of the methods foridentifying proteins in the present invention.

Protein sequencing: N-terminal sequencing aids in the identification ofunknown proteins, confirm recombinant protein identity and fidelity(reading frame, translation start point, etc.), aid the interpretationof NMR and crystallographic data, demonstrate degrees of identitybetween proteins, or provide data for the design of synthetic peptidesfor antibody generation, etc. N-terminal sequencing utilises the Edmandegradative chemistry, sequentially removing amino acid residues fromthe N-terminus of the protein and identifying them by reverse-phaseHPLC. Sensitivity can be at the level of 100 s femtomoles and longsequence reads (20-40 residues) can often be obtained from a few 10 spicomoles of starting material. Pure proteins (>90%) can generate easilyinterpreted data, but insufficiently purified protein mixtures may alsoprovide useful data, subject to rigorous data interpretation.N-terminally modified (especially acetylated) proteins cannot besequenced directly, as the absence of a free primary amino-groupprevents the Edman chemistry. However, limited proteolysis of theblocked protein (e.g. using cyanogen bromide) may allow a mixture ofamino acids to be generated in each cycle of the instrument, which canbe subjected to database analysis in order to interpret meaningfulsequence information. C-terminal sequencing is a post-translationalmodification, affecting the structure and activity of a protein. Variousdisease situations can be associated with impaired protein processingand C-terminal sequencing provides an additional tool for theinvestigation of protein structure and processing mechanisms.

Proteome analyses: Proteomics can be identified primarily by computersearch algorithms that assign sequences to a set of empirically acquiredmass/intensity data which are generated from conducting electrosprayionization (ESI), matrix-assisted laser desorption/ionization(MALDI-TOF), or three-dimensional quadrupole ion traps on the protein ofinterest.

Diagnosis

The identification and analysis of biological substances as disclosedherein has numerous therapeutic and diagnostic applications. Clinicalapplications include, for example, detection of disease, distinguishingdisease states to inform prognosis, selection of therapy, and/orprediction of therapeutic response, disease staging, identification ofdisease processes, prediction of efficacy of therapy, monitoring ofpatients trajectories (e.g., prior to onset of disease), prediction ofadverse response, monitoring of therapy associated with efficacy andtoxicity, and detection of recurrence.

Measuring a concentration of the biological substance can aid in thediagnosis of a course of a disease. For example, the diabetic state of apatient who was previously diagnosed with diabetes can be determined bymonitoring the nasal secretions of the patient for insulin. A biologicalsubstance, for example, growth factor may be one that is specific forthe patient's specific disease. Alternatively, a panel of two or morespecific or non-specific growth factors may be monitored. Theconcentrations of either an individual factor or several factors, in thebiological sample of the patient may be affected by the disease.

The presence or increase or decrease of biological substances'concentration allows the physician or veterinarian to predict the courseof the disease or the efficacy of treatment regimes. If, for example, apatient who had a certain type of disease, which was treated,subsequently exhibits an increase in the concentration of biologicalsubstances that is associated with that disease, the physician orveterinarian can predict that the patient may have progression of thedisease in the future or predict a higher risk of fatality in thepatient. In addition, the amount of biological substances may bepredictive of the outcome of the patient, e.g., how well certainchemotherapeutic agents may act.

One aspect of the present invention is a method of diagnosing a diseaseby obtaining a specimen of nasal secretion, detecting a biologicalsubstance in the specimen, and diagnosing the disease wherein thediagnosis is based on the detection of the biological substance, andwherein the biological substance is not related to a respiratorydisease. In one embodiment leprosy is diagnosed by detection ofantibodies against leprosy causing pathogen for example, mycobacteriumleprae. In one embodiment hepatitis, such as hepatitis A, B, C, D, E,and G, is diagnosed by detection of antibodies against hepatitis causingvirus. In some embodiments, the biological substances include insulin orinsulin receptors for a diagnosis of diabetes. In some embodiments, thebiological substance is p53 for a diagnosis of cancer.

Some embodiments of the invention include diagnosing diabetes bydetecting insulin or insulin receptor in the nasal specimen. Table 6depicts detection and measurement of human insulin concentration innasal mucus as compared to insulin concentration in blood plasma andsaliva. Table 7 depicts the detection and measurement of human insulinreceptor concentration in nasal mucus as compared to the insulinreceptor concentration in plasma and saliva. The appearance of insulinor insulin receptors in nasal mucus refects either their synthesis innasal serous glands or response to a physiological and/or pathologicalphenomena. The presence of insulin or insulin receptors in nasal mucusoffers a non-invasive method for the diagnosis of diabetes and otherdisorders of carbohydrate metabolism.

Some embodiments of the invention include diagnosing cancer by detectingcaspase in the nasal specimen. Cysteine-dependent aspartate-specificproteases (caspases) are a family of proteases that cleave theirsubstrates at aspartic acid (D)-X bonds. 14 mammalian caspases have beenidentified. Caspase-2, -3, -6, -7, -8, -9 and -10 are major players inthe execution phase of apoptosis, whereas caspase-1, -4, -5, and -11 areinvolved in cytokine processing associated with inflammation. Caspase 3,also known as CPP32, cleaves and activates a variety of proteins such assterol regulatory element binding proteins (SREBPs). Caspase-3 alsocleaves poly (ADP-ribose) polymerase (PARP) at the onset of apoptosisand amyloid β precursory protein (APP) which is associated with neuronaldeath in Alzheimer's disease. Caspase 3 is activated by graszyne β,ADAF-1, caspase 9 and caspases 6, 8 and 10. This substance is one of theapoptotic substances found during the apoptotic process. Table 10illustrates a comparison between the detection of caspase 3 in nasalmucus as well as saliva. The presence of caspase in nasal mucus is about13% of that in saliva and reflects the magnitude of the apoptoticprocess. The presence of caspase in nasal mucus indicates the activityof cellular death in nasal mucus and shows that cancer can be diagnosedby detecting caspase in the nasal specimen.

Some embodiments of the invention include diagnosing cancer, taste lossor smell loss by detecting tumor necrosis factor α (TNFα) in the nasalspecimen. TNFα is a 17 KD cleavage product mediated by TNFα convertingenzyme which interacts with two distinct TNFα receptors (I, II) on thecell surface. TNFα is upregulated in many pathological processesinvolving inflammation and oncological processes such as rheumatoidarthritis, refractory bronchial asthma, liver disease, cancer and inpatients with taste and smell loss. It is also called cachectin and isproduced by many normal and tumor cells in response to a wide variety ofstimuli including viruses, bacteria, parasites, cytokines and mitogens.Both the transmembrane and the soluble secreted forms of TNFα arebiologically active. TNFα is an extremely pleotrophic cytokine due tothe ubiquity of its receptors, its ability to activate multiple signaltransduction pathways and its ability to induce or suppress theexpression a large number of genes.

Detecting the levels of TNFα in nasal mucus as disclosed herein makesthe diagnosis of a disease possible on a clinical basis since obtainingcellular diagnosis through tissue biopsy can not only be invasive butalso can be difficult and at times dangerous. Table 11 illustratesdetection and measurement of TNFα in nasal mucus and saliva in 75subjects. Results indicate that TNFα in nasal mucus is about 30 timeshigher than in saliva. These data suggest that various cancers can bediagnosed by measurements of TNFα in nasal mucus and their treatment canbe monitored by following its concentration in nasal mucus. Since levelsof TNFα may also reflect the inflammatory aspects of disease processesinducing it, use of anti TNFα drugs through nasal administration reflecta method of treating these various disease processes. Concentrations ofTNFα in nasal mucus in patients with smell loss can be greater than forexample, 5000 times that found in normal subjects thereby reflecting itsfunction as a “death protein” indicator of excessive apoptosis as in itsrole in cancer.

Monitoring the levels of TNFα in nasal mucus may help in diagnosis ofarterial venous malfunction (AVM). AVM is normally diagnosed by MRI butdetection and measurement of TNFα in nasal mucus provides a non-invasivemethod of diagnosing AVM. Monitoring the level of TNFα can help indiagnosing the progression or stage of AVM or the susceptibility of thesubject towards AVM.

Some embodiments of the invention include diagnosing cancer, taste lossor smell loss by detecting tumor necrosis factor receptor I (TNFR I) inthe nasal specimen. TNF receptor I (TNFRI) is one of the two cellularreceptors upon which TNFα operates. It is one of the prototypic membersof the TNF receptor super family members designated TNFRSF I α. Indisease processes associated with increased TNFα activity TNFR I may beupregulated. Its presence in nasal mucus can reflect the activity ofmany inflammatory, oncological and other pathological processes,including taste and smell dysfunction. Table 12 illustrates detectionand measurement of TNFR I in 47 subjects. Results indicate that TNFR Iin nasal mucus is about 16 times its concentration in saliva and itsconcentration is significantly increased over that found in plasma, redblood cells, or urine. Thus the detection of TNFR I in the nasalspecimen can be used to establish clinical diagnoses of excessiveapoptosis and can be used as a treatment modality in inhibitingpathological apoptosis.

TNFR II is the other of the two cellular receptors upon which TNFαoperates. TNFR II is one of the high-affinity receptors for TNFα and isone of the prototypic members of the TNF super family members designatedTNFSF I β. TNFR II may be upregulated in many inflammatory andoncological disease processes. It may also be solubilized and haveproperties similar to TNFR I. Table 13 illustrates detection andmeasurement of TNFR II in 47 subjects. Results indicate that TNFR II innasal mucus is about 24 times its concentration in saliva and itsconcentration in nasal mucus is significantly higher than found inplasma, rbcs, or urine. The results reflect that detection of TNFR II innasal specimen can provide a non invasive method of diagnosing variouspathological processes related to TNFR II.

TNF related apoptosis-inducing ligand (TRAIL) is also known as apo-2ligand and TNFSF-10. It is a Type II transmembrane protein with acarboxy terminal extracellular domain that exhibits homology to otherTNF super family members. Among TNF super family members TRAIL is themost homologous Fas ligand, sharing 28% amino acid sequence identity intheir extracellular domain. Human TRAIL shares 65% amino acid sequenceidentity with mouse TRAIL. TRAIL reflects the terminal protein in theapoptotic sequence. Table 14 in the examples illustrates detection andmeasurement of TRAIL in saliva and nasal mucus in normal subjects and inpatients with smell loss. Results indicate that TRAIL in nasal mucus isabout 5 times higher than in saliva and both are significantly higherthan in blood, rbcs or urine. The results reflect that detection ofTRAIL in nasal mucus can provide a non invasive method of diagnosingvarious diseases related to TRAIL. The methods of the present inventioninclude treatment of diseases by modulating these elevatedconcentrations by use of anti-TRAIL drugs or agents. In someembodiments, the treatment is preferably by nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting interleukin in the nasal specimen. Interleukin 2 (IL2) is aT-cell growth factor that is produced by T-cells following activation bymitogins or antigens and it stimulate growth and differentiation of βcells, natural killer (NK) cells, lymphocyte killer (LAK) cells,monocytes/macrophages and oligodendrocytes. At the amino acid sequencelevel there can be about 50-90% homology between species. Interleukin 3(IL 3), also known as mast cell growth factor, is a pleitrophic factorproduced primarily by activated T cells. It can stimulate proliferationand differentiation of pluripotent hematopoetic stem cells as well asvarious lineage committed progenitors. Mature human and mouse IL 3 shareabout 29% amino acid sequence homology. Table 18 in the examplesillustrates detection and measurement of IL 3 in both human saliva andnasal mucus. Levels of IL 3 in nasal mucus were found to be about ½ theconcentration in saliva but both levels were higher than that found inplasma, rbcs or urine. IL 3 present in nasal mucus provides a noninvasive method of diagnosing various diseases related to IL3. Themethods of the present invention include treatment of diseases bymodulating the concentration of IL3 with drugs. In some embodiments, thetreatment is by nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting endostatin in the nasal specimen. Endostatin is a 20 KD Gterminal fragment of collagen XVII. Its function is as an antiangiogenicsubstance or angiogenic antagonist. It is a naturally occurring proteinwhich has been used as an anti cancer agent to inhibit blood vesselgrowth and spread of any form of cancer. Table 19 in the examplesillustrates detection and measurement of endostatin in plasma, urine,saliva and nasal mucus in 15 subjects. Endostatin levels in nasal mucuswere 5 times higher than in saliva but 7% that found in plasma. On thebasis of endostatin/protein, levels of nasal mucus are about 14% thatfound in plasma. Presence of endostatin in nasal mucus indicates anon-invasive method of detection of endostatin in nasal mucus and itsuse in diagnosing various diseases. The methods of the present inventioninclude treatment of diseases by modulating the concentration ofendostatin with drugs. In some embodiments, the treatment is by nasaladministration.

Some embodiments of the invention include diagnosing disease bydetecting erythropoietin in the nasal specimen. Erythropoietin (EPO) isa 30 KD glycosylated protein produced primarily by the kidney. It is theprincipal factor that regulates erythropoesis. Production of EPO by thekidney cell is increased in response to hyposmia or anemia. The cDNA forEPO has been cloned from many species. Mature proteins from variousspecies are highly conserved exhibiting greater than 80% amino acidsequence homology. Table 20 in the examples illustrates detection andmeasurement of EPO in plasma, urine, saliva and nasal mucus. EPO was notfound in urine or saliva. The level of EPO in nasal mucus was found tobe between 1.1 and 4.5 times higher than in plasma. Presence of EPO innasal mucus illustrates a non-invasive method of detection of EPO innasal mucus and its use in diagnosing various diseases. The diagnosiscan further lead to treatment of disease by modulating the concentrationof EPO with drugs. In some embodiments, the treatment is by nasaladministration.

Some embodiments of the invention include diagnosing disease bydetecting bone morphogenic protein in the nasal specimen. Bonemorphogenic protein BMP I, also known as procollagen C-proteinase (PCP)is a zinc protease of the astacin family. BMP I/PCP plays a key role information of extracellular matrix (KCM) by connecting precursor proteinsinto their mature and functional form. Precursor proteins identified assubstrates for BMP I/PCP include collagens, biglycan, laminin S, dentinmatrix protein I and lysyl oxidase. Table 21 in the examples illustratesdetection and measurement of BMP I in plasma, urine, saliva and nasalmucus in 20 subjects. BMP I was found in plasma but not in urine, salivaor nasal mucus.

Some embodiments of the invention include diagnosing disease bydetecting brain-derived neurotrophic factor in the nasal specimen.Brain-derived neurotrophic factor (BDNF) is a member of the NGF familyof neurotrophic factors, BDNF, NGF, NT-3 and NT 4/5. BDNF is requiredfor differentiation and survival of specific subpopulations in bothcentral and peripheral nervous systems. High levels of BDNF expressionhave been found in hippocampus, cerebellum, fetal eye and placenta.Table 22 in the examples illustrates detection and measurement of BDNFin plasma, urine, saliva and nasal mucus in 20 subjects. BDNF was foundin plasma and nasal mucus but not in urine or saliva. Levels of BDNF inplasma were higher than in nasal mucus. The results indicate that nasalmucus is a repository of the family of nerve growth factors and theconcentration of BDNF as shown in Table 22, may help understand bothphysiology and pathology of neurotrophic factors related to growth andhomeostasis of cells in the nasal cavity as well as reporting on thepresence of these factors in the systemic circulation. Presence of BDNFin nasal mucus illustrates a non-invasive method of detection of BDNF innasal mucus and its use in diagnosing various diseases. The diagnosiscan further lead to treatment of diseases by modulating theconcentration of BDNF with drugs. In some embodiments, the treatment isby nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting ciliary neurotrophic factor in the nasal specimen. Ciliaryneurotrophic factor (CNTF) is structurally related to IL-6, IL-11, L1F,CLC and OSM. CNTF is a trophic factor for embryonic chick ciliaryparasympathetic neurons in culture. CNTF is also a survival factor foradditional numerous cell types including dorsal root ganglion sensoryneurons, sympathetic ganglion neurons, embryonic motor neurons, majorpelvic ganglion neurons and hippocampal neurons. Table 23 in theexamples illustrates detection and measurement of CNTF in plasma, urine,saliva and nasal mucus in 19 subjects. Levels of CNTF in plasma andnasal mucus were found to be similar but lower in saliva. Presence ofCNTF in nasal mucus illustrates a non-invasive method of detection ofCNTF in nasal mucus and its use in diagnosing various diseases. Thediagnosis can further lead to treatment of diseases by modulating theconcentration of CTNF with drugs. In some embodiments, the treatment isby nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting granulocyte macrophage growth factor in the nasal specimen.Granulocyte macrophage growth factor (GM-CSF) is a 22 KD mononerichematopoetic cytokine that is characterized as a growth factor thatsupports the in vitro colony formation of granulocyte macrophageprogenitors. It is produced by a number of different cell typesincluding activated T cells, B cells, macrophages, mast cells,endothelial cells and fibroblasts, in response to cytokines or immuneand inflammatory stimuli. GM-CSF is species specific. Table 24 in theexamples illustrates detection and measurement of GM-CSF in plasma,urine, saliva and nasal mucus in 16 subjects. The results provide anon-invasive method for the detection of GM-CSF in nasal mucus. Levelsin nasal mucus were found to be over 6 times that found in plasma. Thedetection of GM-CSF in nasal mucus provides a non invasive method ofdiagnosing various diseases related to GM-CTF. The methods of thepresent invention include treatment of diseases by modulating theconcentrations of GM-CSF by use of drugs. In some embodiments, thetreatment is by nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting hepatocyte growth factor in the nasal specimen. Hepatocytegrowth factor (HGF), also known as hepatopoeitin A, is a mitogenicprotein for a variety of cell types including endothelial and epithelialcells, melanocytes and keratinocytes. It is identical to scatter factor,a fibroblast-derived soluble factor that promotes the dissociation ofepithelial and vascular endothelial cell colonies in monolayes culturesby stimulating cell migration. Table 25 in the examples illustratesdetection and measurement of HGF in plasma, urine, saliva and nasalmucus in 17 subjects. Concentrations of HGF in nasal mucus were found tobe higher than that found in either plasma or urine. These resultssuggest that HGF may be synthesized in the serous glands of the nose fora specific mechanism involved with nasal homeostasis as well as amechanism involved with systemic cell migration. The results provide anon-invasive method for the detection of HGF in nasal mucus. Thedetection of HGF in nasal mucus provides a non invasive method ofdiagnosing various diseases related to human physiology and pathology.The diagnosis further leads to a treatment of diseases by modulating theconcentrations of HGF by use of drugs.

Some embodiments of the invention include diagnosing disease bydetecting platelet derived growth factor in the nasal specimen. Plateletderived growth factor (PDGF) family is a group of disulfide-linkeddimeric proteins which act mainly on connective tissue. This family mayconsist of four homodimeric proteins, PDGF-AA, PDGF-BB, PDGF-CC andPDGF-DD and one heterodimeric protein, PDGF-AB. The technique of ELISAmeasurement used is associated with the ability of PDGF to stimulateincorporation of 3H-thymidine in quiescent NRGR-3 T 3 fibroblastis.Table 26 in the examples illustrates detection and measurement of PDGFin human plasma, urine, saliva and nasal mucus in 18 subjects.Concentrations of PDGF expressed per mg protein were found to be higherin saliva and nasal mucus than in plasma. These results suggest thatPDGF may be synthesized in the serous glands of the nose for a specificmechanism involved with nasal homeostasis. The results provide anon-invasive method for the detection of PDGF in nasal mucus. Thedetection of PDGF in nasal mucus provides a non invasive method ofdiagnosing various diseases related to human physiology and pathology.The diagnosis can provide treatment of diseases by modulating theconcentrations of PDGF by use of drugs.

Some embodiments of the invention include diagnosing taste loss or smellloss by detecting carbonic anhydrase in the nasal specimen. Carbonicanhydrase is a zinc-containing enzyme and at least twenty carbonicanhydrase variants, called “isozymes” have been identified. CarbonicAnhydrase VI (CA VI) is a 36 KD zinc metalloglycoprotein. Its synthesisin nasal mucus may take place in nasal serous glands (in the oralparotid glands). It can act as a taste bud growth factor in the oralcavity and as an olfactory receptor growth factor in the nasal cavity.It can also act on taste bud and olfactory receptor stem cells to inducegrowth and development of the entire panoply of cell types for the tastebuds and olfactory epithelium. Its decreased synthesis may induce bothloss of taste and smell. Its resumed synthesis may return cell growth tonormal. Treatment which increases synthesis of CA VI may involve severalcomplex processes including increasing zinc-cofactor concentration.Administration of zinc ion to some patients who are either zincdeficient or who may have metabolic processes which inhibit zincincorporation into the protein, may have their taste and smell functionimproved through this treatment. Since the carbohydrates in this proteinare part of its function, any process that repairs glycoproteinincorporation into this protein may also therapeutically be effective inrestoring taste and smell function.

Table 27 in the examples illustrates decrease in CA VI in patients withsmell and taste loss. Table 28 in the examples illustrates loss of smellfunction by disease etiology with respect to measurements of CA VIconcentration in nasal mucus. Results indicate that patients with postinfluenza hyposmia hypogeusia (PIHH), allergic rhinitis and postanesthesia have significantly decreased CA VI concentrations in nasalmucus. These results provide a method for the detection and measurementof CA VI in nasal mucus as an index of smell and taste loss and itscontinual measurement during treatment of these disorders in order tomonitor efficacy of therapy. The detection of CA VI in nasal mucusprovides a non invasive method of diagnosing various diseases related tohuman physiology and pathology. The diagnosis can further lead totreatment of diseases by modulating the concentrations of CA VI by useof drugs

Some embodiments of the invention include diagnosing a disease bydetecting cAMP and cGMP in the nasal specimen. Table 29 in the examplesillustrates detection and measurement of cAMP and cGMP in saliva and innasal mucus in normal subjects. Table 30 in the examples illustratescomparison of the measurement of cAMP and cGMP in normal subjects withthe patients with taste and smell loss. Results indicated that patientswith smell loss had decreased levels of cAMP in their nasal mucus. Theseresults indicate that cAMP in nasal mucus can be an index of smell lossand that its secretion may be inhibited in smell loss. The resultsprovide a non-invasive method for the detection of cAMP and cGMP innasal mucus.

Table 31 in the examples illustrates detection and measurement of cAMPand cGMP secretion in nasal mucus in patients with graded severity ofsmell loss (anosmia<Type I hyposmia<Type II hyposmia from most severe toleast severe). Data indicates that as degree of smell loss increased,levels of cAMP in nasal mucus decreased. These data confirm therelationship between cAMP secretion in nasal mucus and degree of smellloss. Results also indicate that there was less significant differencebetween cGMP in nasal mucus in normal subjects or in patients withhyposmia. However, the concentration of cGMP in saliva is essentiallysimilar to that of cAMP, phenomena different from that observed in othertissues.

The ability to smell and, in part, the ability to taste or to obtainflavor from food is regulated by the olfactory nerve system. Theolfactory nerve system is complex and interconnected with severalsystems in the brain. Olfactory receptors located in the nose arespecialized bipolar neurons with cilia protruding into the mucouscovering the epithelium. The axons of the bipolar neurons are packedinto bundles that form connections in the olfactory bulb in the brain.The olfactory bulbs contain a rich supply of neurotransmitters andneuromodulators. Chemosensory dysfunctions are usually described by thefollowing terms: ageusia (absence of taste), hypogeusia, (diminishedsensitivity of taste), dysgeusia (distortion of normal taste), anosmia(absence of smell), hyposmia (diminished sense of smell), and dysosmia(distortion of normal smell).

Treatment with drugs which increase cAMP secretion (e.g., thephosphodiesterase theophylline or cilostazol) increases nasal mucus cAMPconcentration and are associated with increases in smell function. Thus,cAMP measurements are critical to monitor both loss of smell functionand changes in smell function following treatment. The detection of cAMPand cGMP in nasal mucus provides a non invasive method of diagnosingvarious diseases related to human physiology and pathology. The methodsof the present invention include treatment of diseases by modulating theconcentrations of cAMP and cGMP by use of drugs or agents. The method oftreatment is preferably by nasal administration.

Some embodiments of the invention include diagnosing a disease bydetecting nitric oxide in the nasal specimen. Nitric Oxide (NO) is apletrophic-signaling molecule implicated in diverse biological processesincluding inhibition of platelet aggregation, regulation ofneurotransmission, vasodilation, immune responses and inflammation. NOis synthesized from arginine and O₂ by three nitric oxide synthase(NOS), enzymes endothelial NOS (eNOS), neuronal NOS (nNOS), andinducible NOS (iNOS). Each enzyme isoform is expressed in a variety oftissues and cell types. While eNOS and nNOS generally exhibitconstitutive expression and are involved in physiological signaling andcellular maintenance functions, iNOS expression may be induced byinflammatory stimuli and may be associated with both normal andpathological immune responses. Table 32 in the examples illustratesdetection and measurement of NO in human saliva and nasal mucus. NO wasfound to be present is in both saliva and nasal mucus and its meanconcentration in saliva were 21% lower in patients than in normalsubjects whereas in nasal mucus mean levels were 25% lower in patients.Treatment which increases cAMP in nasal mucus and improves smellfunction may be mirrored by increases in nasal mucus NO.

Some embodiments of the invention include diagnosing a disease bydetecting insulin-like growth factor I in the nasal specimen.Insulin-like growth factor I (IGF 1), also known as somatomedin Cbelongs to the family of insulin-like growth factors that arestructurally homologous to proinsulin. IGF 1 is a potent mitogenicfactor that mediates growth-promoting activities of growth hormonepostnatally. IGF 1 also promotes growth during embryonic growth anddifferentiation. Table 34 in the examples illustrates detection andmeasurement of IGF 1 in human saliva and nasal mucus in 26 subjects.Results show that IGF 1 concentration in nasal mucus was significantlygreater than in saliva. Results indicate that the measurement of nasalmucus IGF 1 can be used as an index of human physiology and pathology.The detection of IGF 1 in nasal mucus provides a non invasive method ofdiagnosing various diseases related to human physiology and pathology.The diagnosis can further help in treatment of diseases by modulatingthe concentrations of IGF 1 by use of drugs.

Some embodiments of the invention include diagnosing a disease bydetecting endoglin in the nasal specimen. Endoglin, also known as CD105, is a type 1 integral membrane glycoprotein and is an accessoryreceptor for TGF-β super family ligands. Endoglin is expressed onvascular endothelial cells, chrondrocytes and syncytiotrophoblasts ofterm placenta. It is also found on activated monocytes, mesenchymol stemcells and leukemic cells of lymphoid and myeloid lineages. Table 39illustrates detection and measurement of endoglin in the nasal mucus.Results indicate that the measurement of nasal mucus endoglin can beused as an index of human physiology and pathology. The detection ofendoglin in nasal mucus provides a non invasive method of diagnosingvarious diseases related to human physiology and pathology.

Some embodiments of the invention include diagnosing a disease bydetecting fibroblast growth factor (FGF) in the nasal specimen. FGFacidic is a member of the FGF family of mitogenic peptides. Unlike othermembers of the family, it lacks signal peptides. FGF is apparentlysecreted by mechanisms other than the classical protein secretionpathways. There are approximately 23 distinct members of this family.The nucleotide sequence of human FGF acidic is well known and it is a155 amino acid protein. FGF mediates cellular responses by binding toand activating a family of four receptor tyrosine kinases. FGF isinvolved in wound healing as it binds heparin. It promotes endothelialcell proliferation by physical organization of endothelial cells intotubes. It promotes angiogenesis and stimulates the proliferation offibroblasts that give rise to granulation tissue. It is a more potentangiogenic factor than either VFGF or PDGF. It acts on PC 12 cells;these cells also respond to NGF in a similar manner. Low levels of FGFhave been found in blood of patients with depression. Acidic FGF wasmeasured in blood plasma, urine, saliva and nasal mucus in 13 subjects.No FGF was found in any sample of plasma, urine or saliva. FGF wasmeasured in three samples of the nasal mucus or in 23 percent of thesubjects. Values ranged from 8-44 pg/ml with a mean±SEM of 24±13 pg/ml.These results suggest that FGF is present in nasal mucus and may be partof a feedback mechanism involving nasal cavity and brain since FGF issynthesized in the brain.

The diagnosis of the disease as disclosed herein can be used to enableor assist in the pharmaceutical drug development process for therapeuticagents. The analysis can be used to diagnose disease for patientsenrolling in a clinical trail. The diagnosis can indicate the state ofthe disease of patients undergoing treatment in clinical trials, andshow changes in the state during the treatment. The diagnosis candemonstrate the efficacy of a treatment, and can be used to stratifypatients according to their responses to various therapies.

The methods of the present invention can be used to evaluate theefficacy of treatments over time. For example, sample of nasalsecretions can be obtained from a patient over a period of time as thepatient is undergoing treatment. The DNA from the different samples canbe compared to each other to determine the efficacy of the treatment.Also, the methods described herein can be used to compare the efficaciesof different therapies and/or responses to one or more treatments indifferent populations (e.g., different age groups, ethnicities, familyhistories, etc.).

In preferred embodiment, at least one step of the methods of the presentinvention is performed using a computer as depicted in FIG. 2. FIG. 2illustrates a computer for implementing selected operations associatedwith the methods of the present invention. The computer 200 includes acentral processing unit 201 connected to a set of input/output devices202 via a system bus 203. The input/output devices 202 may include akeyboard, mouse, scanner, data port, video monitor, liquid crystaldisplay, printer, and the like. A memory 204 in the form of primaryand/or secondary memory is also connected to the system bus 203. Thesecomponents of FIG. 2 characterize a standard computer. This standardcomputer is programmed in accordance with the invention. In particular,the computer 200 can be programmed to perform various operations of themethods of the present invention.

The memory 204 of the computer 200 may store a detection/diagnosismodule 205. In other words, the detection/diagnosis module 205 canperform the operations associated with step 102, 103, and 104 of FIG. 1.The term “detection/diagnosis module” used herein includes, but notlimited to, analyzing one or more biological substances, identifying thebiological substance, and diagnosing the disease after theidentification. The executable code of the detection/diagnosis module205 may utilize any number of numerical techniques to perform thediagnosis.

Examples of Biological Substances

Various substances that can be diagnosed by the methods of the presentinvention include, by way of example only, proteins, carbohydrates,lipids, hormones (e.g., leptin, ghrelin) in control of appetite,cholesterol and other lipids and lipid carrying proteins in control oflipid metabolism, growth factors (e.g., hepatic growth factor,granulocyte colony growth factor, brain derived neurotrophic factor),liver enzymes (SGOT, SGPT) therapeutic and recreational drugs of abuse,trace metals [either excess as in toxicity (e.g., lead, mercury,arsenic) or in deficiency diseases involving zinc, copper, magnesium]and most other substances found in plasma, erythrocytes, urine andsaliva. Each metabolite in nasal mucus may reflect both physiologicaland pathological changes in human body metabolism specific to eachmetabolite and may reflect the manner in which nasal mucus providesinformation both on human body metabolism such as provided by plasma,erythrocytes, urine and saliva or information relatively unique to nasalmucus.

The methods of the present invention include PCR to enable detectionand/or characterization of specific nucleic acid sequences associatedwith infectious diseases, genetic disorders or cellular disorders.Various infectious diseases can be diagnosed by the presence in clinicalsamples of specific DNA sequences characteristic of the causativemicroorganism.

Infectious organisms may comprise viruses, (e.g., single stranded RNAviruses, single stranded DNA viruses, human immunodeficiency virus(HIV), hepatitis A, B, and C virus, herpes simplex virus (HSV),cytomegalovirus (CMV) Epstein-Barr virus (EBV), human papilloma virus(HPV)), parasites (e.g., protozoan and metazoan pathogens such asPlasmodia species, Leishmania species, Schistosoma species, Trypanosomaspecies), bacteria (e.g., Mycobacteria, M. tuberculosis, Salmonella,Chlamydia, Neisseria, Streptococci, E. coli, Staphylococci, C. psittaciand C. pecorum), fungi (e.g., Acremonium; Absidia (e.g., Absidiacorymbifera). Aspergillus (e.g., Aspergillus clavatus, Aspergillusflavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger,Aspergillus terreus, Aspergillus versicolor, etc), Blastomyces (e.g.,Blastomyces dermatitidis, etc), Candida (e.g., Candida albicans, Candidaglabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candidaparapsilosis, Candida stellatoidea, Candida tropicalis, Candida utilis,etc.), Cladosporium (e.g., Cladosporium trichoides, etc), Coccidioides(e.g., Coccidioides immitis, etc), Cryptococcus (e.g., Cryptococcusneoformans, etc), Cunninghamella (e.g., Cunninghamella elegans, etc),Dermatophyte, Exophiala (e.g., Exophiala dermatitidis, Exophialaspinifera, etc), Epidermophyton (e.g., Epidermophyton floccosum, etc),Fonsecaea (e.g., Fonsecaea pedrosoi, etc), Fusarium (e.g., Fusariumsolani, etc), Geotrichum (e.g., Geotrichum candiddum, etc), Histoplasma(e.g., Histoplasma capsulatum var. capsulatum, etc), Malassezia (e.g.,Malassezia furfur, etc), Microsporum (e.g., Microsporum canis,Microsporum gypseum, etc), Mucor, Paracoccidioides (e.g.,Paracoccidioides brasiliensis, etc), Penicillium (e.g., Penicilliummarneffei, etc), Phialophora, Pneumocystis (e.g., Pneumocystis carinii,etc), Pseudallescheria (e.g., Pseudallescheria boydii, etc), Rhizopus(e.g., Rhizopus microsporus var. rhizopodiformis, Rhizopus oryzae, etc),Saccharomyces (e.g., Saccharomyces cerevisiae, etc), Scopulariopsis,Sporothrix (e.g., Sporothrix schenckii, etc), Trichophyton (e.g.,Trichophyton mentagrophytes, Trichophyton rubrum, etc), Trichosporon(e.g., Trichosporon asahii, Trichosporon cutaneum, etc).), Pneumocystiscarinii, and prions.

Other examples of biological substances, includes, but is not limitedto, colony stimulating factors (1, 2, 3, GM, α, β, γ, and the like), Bcell factors (B cell growth factor and the like), T cell factors,protein A, suppressive factor of allergy, suppressor factors, cytotoxicglycoprotein, immunocytotoxic agents, immunotoxins, lymphotoxins,cachectin, oncostatins, tumor inhibitory factors, albumin,α-1-antitrypsin, apolipoprotein, erythroid potentiating factors,erythropoietin, factor VII, factor VIII(c), factor IX, hemopoietin-1,kidney plasminogen activator, tissue plasminogen activator, urokinase,pro-urokinase, streptokinase, lipocortin, lipomodulin, macrocortin, lungsurfactant protein, protein C, protein 5, C-reactive protein, renininhibitors, collagenase inhibitors, superoxide dismutase, growthhormone, osteogenic growth factors, atrial naturetic factor, auriculin,atriopeptin, bone morphogenic protein, calcitonin, calcitonin precursor,calcitonin gene-related peptide, cartilage inducing factor, connectivetissue activator protein, fertility hormones (follicle stimulatinghormone, luteinizing hormone, human chorionic gonadotropin), growthhormone releasing factor, osteogenic protein, insulin, proinsulin, nervegrowth factor, parathyroid hormone and analogues, parathyroid hormoneantagonists, relaxin, secretin, somatomedin C, somatostatin andsomatostatin analogues, inhibin, adrenocoricotrophic hormone, glucagon,vasoactive intestinal polypeptide, gastric inhibitory peptide, motilin,cholecystokinin, pancreatic polypeptide, gastrin releasing peptide,corticotropin releasing factor, thyroid stimulating hormone, growthinhibitory factors, vaccine antigens including antigens of HTLV-I, II,HTLV-III/LAV/HIV (AIDS virus), cytomegalovirus, hepatitis A, B, andnon-A/non-B, herpes simplex virus-I, herpes simplex virus II, malaria,pseudorabies, retroviruses, feline leukemia virus, bovine leukemiavirus, transmissible gastroenteritis virus, infectious bovinerhinotracheitis, parainfluenza, influenza, rotaviruses, respiratorysyncytial virus, varicella zoster virus, epstein-barr virus, pertussis,and anti-infective antibodies including monoclonal and polyclonalantibodies to gram negative bacteria, pseudomonas, endotoxin, tetanustoxin, and other bacterial or viral or other infectious organisms.

In addition to naturally-occurring allelic forms of growth inhibitoryfactor, the present invention also embraces other inhibitory factorproducts such as polypeptide analogs of inhibitory factor. Such analogsinclude fragments of inhibitory factor. Other examples of biologicalsubstances, includes, substances that are associated with cancer (eitheractive or remission) and/or with reaction to transplantation (eithertissue acceptance or rejection).

Examples of Diseases

Without limiting the scope of the present invention, the examples ofsome of the diseases which can be diagnosed by detecting the biologicalsubstance, is provided herein. However, these examples are not intendedto limit the scope of the invention. The disease as provided hereininclude, infections, hematological disorders, oncological disorders,endocronological disorders, metabolic disorders, immunologicaldisorders, neurological disorders, vascular disorders, mast celldisorders, psychiatric disorders, neoplastic disorders, nutritionaldisorders, post irradiation disorders, and changes in the trace metalmetabolism.

Infectious diseases include acute and chronic parasitic and/orinfectious diseases from bacterial, viral or fungal sources, but are notlimited to, single or multiple cutaneous lesions, mucosal disease,chagas' disease, toxoplasmosis, leishmaniasis, trypanosomiasis,shistosomiasis, cryptosporidiosis, mycobacterium avium infections,leprosy, dengue, yellow fever, inner ear infections, urinary tractinfections, bacterial endocarditis, osteomyelitis, h. pylori associatedulcers, antibiotic associated colitis, sexually transmitted diseases,malaria, rheumatoid arthritis, inflammatory bowel disease, interstitialcystitis, fibromyalgia, autonomic nervous dysfunction, pyodermagangrenosum, chronic fatigue, chronic fatigue syndrome, sepsis syndrome,cachexia, circulatory collapse and shock resulting from acute or chronicbacterial infection, AIDS (including symptoms of cachexia, autoimmunedisorders, AIDS dementia complex and infections), wegnersgranulomatosis, aneurysms, hemorrhoids, sarcoidosis, chronicinflammatory bowel disease, Crohn's disease, vascular inflammatorypathologies, such as, but not limited to, disseminated intravascularcoagulation, atherosclerosis, and Kawasaki's pathology, inflammatorydiseases such as coronary artery disease, hypertension, stroke, asthma,chronic hepatitis, multiple sclerosis, peripheral neuropathy, chronicvascular headaches (including migraines, cluster headaches, and tensionheadaches), demyelinating diseases, such as multiple sclerosis and acutetransverse myelitis, extrapyramidal and cerebellar disorders, such aslesions of the corticospinal system, disorders of the basal ganglia orcerebellar disorders, hyperkinetic movement disorders such ashuntington's chorea and senile chorea, drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors,hypokinetic movement disorders, such as Parkinson's disease, progressivesupranucleo palsy, cerebellar and spinocerebellar disorders, such asastructural lesions of the cerebellum, spinocerebellar degenerations(spinal ataxia, friedreich's ataxia, cerebellar cortical degenerations,multiple systems degenerations (mencel, Dejerine-Thomas, Shi-Drager, andMachado Joseph)), systemic disorders (Refsum's disease,abetalipoprotemia, ataxia, telangiectasia, and mitochondrialmulti-system disorder), disorders of the motor unit, such as neurogenicmuscular atrophies (anterior horn cell degeneration, such as amyotrophiclateral sclerosis, infantile spinal muscular atrophy and juvenile spinalmuscular atrophy), Alzheimer's disease, Down's Syndrome in middle age,diffuse Lewy body disease, senile dementia of lewy body type,Wernicke-Korsakoff syndrome, chronic alcoholism, Creutzfeldt-Jakobdisease, subacute sclerosing panencephalitis, Hallerrorden-Spatzdisease, and Dementia pugilistica, dermatophytosis (e.g.,trichophytosis, etc), pityriasis versicolor, candidiasis,cryptococcosis, geotrichosis, trichosporosis, aspergillosis,penicilliosis, fusariosis, zygomycosis, sporotrichosis, chromomycosis,coccidioidomycosis, histoplasmosis, blastomycosis,paracoccidioidomycosis, pseudallescheriosis, mycetoma, mycotickeratitis, otomycosis, and pneumocystosis.

Ocular neovascularization, psoriasis, duodenal ulcers etc can also betreated when demonstrated by the diagnostic procedures described herein.Similarly, other diseases (their biological substances in parenthesis),include, but not limited to, neutropenia, gout, dwarfism or congenitalshort stature (growth hormone releasing hormone (GHRH)); congestiveheart disease (atrial natriuretic peptide or factor (ANP or ANF));osteoporosis (parathyroid hormone (PTH)); Paget's disease (calcitonin);accromegally, insulin sparing effects, treatment of long termcomplications of diabetes and treatment of various endocrine secretingtumors (somatostatin); Addison's Disease and Cushing's Syndrome,shipping fever (bovine respiratory syndrome) or ulcers, andstress-induced immunosuppression (corticotrophin releasing factor(CRF)); contraception, fertility control, suppression or interruption ofheat, treatment of ovarian cysts, precocious puberty, prostatichyperplasia and tumors, gynecologic diseases, and termination ofpregnancy (luteinizing hormone-releasing hormone (LHRH)); aplasticanemia, paroxysmal nocturnal hemoglobinurea, chronic myelocyticleukemia, polycythemia vera, essential thrombocythemia, myelofibrosis,myelodysplastic syndrome and acute leukemia; and hematological diseasessuch as megaloblastic anemia, AIDS, multiple myeloma, metastatic cancerof the bone marrow, and drug-induced myelosuppression (hematopoieticstem cell growth factor (SCGF)); body weight disorders, includingobesity, cachexia, and anorexia, and diabetes, neoplasms, andhyperamylinemia (agouti-related protein); impairment of functions,increased ceramide formation, which triggers nitric oxide-mediatedlipotoxicity and lipoapoptosis, obesity and hyperphagia (leptin);hypolipidimia, coronary heart disease, Niemann Pick Disease, Gaucher'sdisease, Batten's syndrome, Farber's lipogranulomatosis, Krabbe'sdisease, metachromic leukodystrophy, Tay-Sach's disease, GM1gangliosidoses, Fabry's disease, cystinosis, aspartylglycosaminuria(lipid profile which includes triglycerides, LDL-cholesterol andHDL-cholesterol), and generalized vascular disease, chronichyperglycemia, obesity, hypertension, atherosclerosis and heart disease,carbohydrate deficient glycoprotein syndrome type 1a, glycogenoses, andgalactosemia (carbohydrates).

Detection of the concentration of the caspases in the nasal secretioncan provide diagnosing a disorder or selection of therapeutic strategiesinvolving, e.g., inappropriate apoptosis and/or excessive cellproliferation, such as an inflammatory disease, a neurodegenerativedisease, cancer, a cardiovascular disease and, any disorder or diseasecharacterized by a gradual and prolonged development of apoptosis.Apoptosis functions in maintaining normal tissue homeostasis in avariety of physiological processes including embryonic development,immune cell regulation, normal cellular turnover and programmed celldeath of cancer cells. Thus, the dysfunction or loss of regulatedapoptosis can lead to a variety of pathological disease states. Forexample, the loss of apoptosis can lead to the pathological accumulationof self-reactive lymphocytes such as occurs in many autoimmune diseases.Inappropriate loss of apoptosis can also lead to the accumulation ofvirally infected cells and of hyperproliferative cells such asneoplastic or tumor cells. Inappropriate activation of apoptosis cancontribute to a variety of diseases such as AIDS, neurodegenerativediseases and ischemic injury.

Dysregulation of apoptosis has been implicated in numerous diseases suchas neurodegenerative disorders including Alzheimer's disease,Parkinson's disease, Huntington's disease, and amyotrophic lateralsclerosis (ALS), cerebellar degeneration, stroke, traumatic braininjury, CNS ischemic reperfusion injury including neonatalhypoxic-ischemic brain injury or myocardial ischemic-reperfusion injury,injury caused by hypoxia, cardiovascular diseases (e.g., myocardialinfarction), especially those which are associated with apoptosis ofendothelial cells, degenerative liver disease, multiple sclerosis,rheumatoid arthritis, hematological disorders including lymphoma,leukemia, aplastic anemia, and myelodysplastic syndrome, osteoporosis,polycystic kidney disease, AIDS, myelodysplastic syndromes, aplasticanemia and baldness. Diseases of the eye include glaucoma, retinitispigmentosa and macular degeneration.

Inflammatory disease states include systemic inflammatory conditions andconditions associated locally with migration and attraction ofmonocytes, leukocytes and/or neutrophils. Inflammation may result frominfection with pathogenic organisms (including gram-positive bacteria,gram-negative bacteria, viruses, fungi, and parasites such as protozoaand helminths), transplant rejection (including rejection of solidorgans such as kidney, liver, heart, lung or cornea, as well asrejection of bone marrow transplants including graft-versus-host disease(GVHD)), or from localized chronic or acute autoimmune or allergicreactions. Autoimmune diseases include acute glomerulonephritis;rheumatoid or reactive arthritis; chronic glomerulonephritis;inflammatory bowel diseases such as Crohn's disease, ulcerative colitisand necrotizing enterocolitis; granulocyte transfusion associatedsyndromes; inflammatory dermatoses such as contact dermatitis, atopicdermatitis, psoriasis; systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, and some forms of diabetes, or anyother autoimmune state where attack by the subject's own immune systemresults in pathologic tissue destruction. Allergic reactions includeallergic asthma, chronic bronchitis, acute and delayed hypersensitivity.Systemic inflammatory disease states include inflammation associatedwith trauma, burns, reperfusion following ischemic events (e.g.thrombotic events in heart, brain, intestines or peripheral vasculature,including myocardial infarction and stroke), sepsis, ARDS or multipleorgan dysfunction syndrome. Inflammatory cell recruitment also occurs inatherosclerotic plaques.

Examples of pathological conditions resulting from increased cellsurvival include cancers such as lymphomas, carcinomas andhormone-dependent tumors (e.g., breast, prostate or ovarian cancer).Abnormal cellular proliferation conditions or cancers that may betreated in either adults or children include solid phasetumors/malignancies, locally advanced tumors, human soft tissuesarcomas, metastatic cancer, including lymphatic metastases, blood cellmalignancies including multiple myeloma, acute and chronic leukemias,and lymphomas, head and neck cancers including mouth cancer, larynxcancer and thyroid cancer, lung cancers including small cell carcinomaand non-small cell cancers, breast cancers including small cellcarcinoma and ductal carcinoma, gastrointestinal cancers includingesophageal cancer, stomach cancer, colon cancer, colorectal cancer andpolyps associated with colorectal neoplasia, pancreatic cancers, livercancer, urologic cancers including bladder cancer and prostate cancer,malignancies of the female genital tract including ovarian carcinoma,uterine (including endometrial) cancers, and solid tumor in the ovarianfollicle, kidney cancers including renal cell carcinoma, brain cancersincluding intrinsic brain tumors, neuroblastoma, astrocytic braintumors, gliomas, metastatic tumor cell invasion in the central nervoussystem, bone cancers including osteomas, skin cancers includingmalignant melanoma, tumor progression of human skin keratinocytes,squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma andKarposi's sarcoma.

Viral infections that may be detected include infections caused byherpesviruses (including CMV, HSV-1, HSV-2, VZV, EBV, HHV-6, HHV-7 andHHV-8), paramyxoviruses (including parainfluenza, mumps, measles, andrespiratory syncytial virus (RSV)), picornaviruses (includingenteroviruses and rhinoviruses), togaviruses, coronaviruses,arenaviruses, bunyaviruses, rhabdoviruses, orthomyxoviruses (includinginfluenza A, B and C viruses), reoviruses (including reoviruses,rotaviruses and orbiviruses), parvoviruses, adenoviruses, hepatitisviruses (including A, B, C, D and E) and retroviruses (including HTLVand HIV). Treatments of both acute and chronic infection arecontemplated.

Adenylyl cyclases are a family of enzymes that catalyze the formation ofAdenosine-3′:5-cyclic monophospate (cAMP) from adenosine-5′-triphosphate(5′ATP), mediate the physiological effects of numerous hormones andneurotransmitters, and belong to a super family of membrane-boundtransporters and channel proteins. Adenosine-3′:5′-cyclic monophosphate(cAMP) is the second messenger involved in signal transduction fornumerous neurotransmitters and hormones, and thus may have an impactupon some of the mediators for smooth muscle cells (SMC) proliferationand migration. Many hormones and other substances may activate cAMP andmay activate the subsequent signaling cascades via their indirectinfluence on adenylyl cyclase. cAMP is a growth factor for neuritegrowth and is involved in development in tissue culture of sympatheticganglion cells similar to the action of NGF. Adenylyl cyclase is agrowth factor which acts on stem cells in taste buds and olfactoryepithelium to induce growth and development of all cell types in tastebuds and olfactory epithelium. cGMP, guanosine 3′, 5″-cyclicmonophosphate is formed by the action of guanylyl cyclase on GTP. cGMPis present at levels typically lower than cAMP in most tissues.Hormones, such as insulin and oxytocin as well as other substancesincluding acetylcholine, serotonin and histamine may increase cGMPlevels. Stimulators of cGMP may include vasodilators and peptides thatrelax smooth muscle.

Adenylyl cyclases also play a role in the disease progression ofCongestive heart failure (CHF). CHF is defined as an abnormal heartfunction resulting in an inadequate cardiac output for metabolic needs.Heart failure is usually not recognized until a more advanced stage ofheart failure which is referred to as congestive heart failure. Onphysical examination, patients with CHF tend to have elevations in heartand respiratory rates, rates (an indication of fluid in the lungs),edema, jugular venous distension, and, in general, enlarged hearts. Themost common cause of CHF is atherosclerosis which causes blockages inthe blood vessels (coronary arteries) that provide blood flow to theheart muscle. Ultimately, such blockages may cause myocardial infarction(death of heart muscle) with subsequent decline in heart function andresultant heart failure.

Fibroproliferative vasculopathy includes restenosis following coronarybypass surgery and PTCA (percutaneous transluminal coronaryangioplasty), allograft arteriosclerosis in chronic allograft rejection,diabetic angiopathy and all forms of common arteriosclerosis. Vascularintimal dysplasia and remodeling are characteristic features of reinjuryfollowing balloon angioplasty, coronary bypass surgery and in chronicallograft rejection. An initial response to vascular injury isinflammatory and involves attraction of lymphocytes, macrophages andthrombocytes to the site of injury and secretion of cytokines,eicosanoids and growth factors. Under the influence of growth factorsand cytokines, smooth muscle cells (SMC) may proliferate and migratefrom the media to the intima and contribute to intimal hyperplasia andstenosis. cAMP has an impact upon some of the key mediators for SMCproliferation and migration.

Glucose is irreversibly oxidized within the cells to produce water andcarbon dioxide. In the presence of a catalyst, especially a carbonicanhydrase enzyme (of which several forms exist, of which the formpresent depends upon the type of tissue cells present), the water andcarbon dioxide may reversibly produce a hydrogen ion and a bicarbonateion. Hydrogen ion produced by carbonic anhydrase enzymes can be actedupon by cytochrome system, which can then be utilized as the energysource of the ion pump that maintains the integrity of the cell membranecomprising and enclosing each cell. It can also be a source of thebrain's electric current. Disruption of the process may causedepolarization of the cell wall membrane, hence sodium (Na), water, andother chemicals can enter the cell in uncontrolled amounts and potassium(K) can exit uncontrollably, leading to the death and destruction of theinvolved cells followed by cellular edema. As this edema progresses, thecell dies. Along with the progressive and gradual death of cells,gliosis may follow resulting in the aging in the brain. The deficiencyof carbonic anhydrase can cause conditions of aging associated with adecreased presence of cell-specific carbonic anhydrase enzymes in thebrain, such as chronic neurodegenerative conditions including dementiasuch as Alzheimer's disease, or showing other forms of dementia orneurodegenerative diseases.

Methods of Treatment

The substances secreted into saliva and nasal mucus act on local oraland nasal tissues, respectively, to induce physiological effects. Thereare several effects of gland secretion at distant sites: (1)endocrine-secretions from a gland and subsequent action at a distantsite, the secretion carried in blood to the distant site; (2)paracrine-secreted substances act at a distant site within the localreach of the fluid; (3) exocrine-secretions from a gland which havedirect local effects, e.g, β-cells in the pancreas which act directly tosecrete insulin in response to local changes in blood glucose. This is aone directional effect, a secretion from the gland, into the biologicalfluid, acting at a distant but local site.

There are feedback mechanisms such that whatever effects the glandsecretion had on its receptor, the receptor also interacted with thesite of secretion. For example, increased glucose induces increasedsecretion of insulin but as insulin secretion increases, insulinreceptor number in liver and pancreas change in response to theincreased insulin secretion. This feedback concept can also beexemplified by brain secretion of peptide hormones which acted as masterfeedback mechanisms to control peripheral hormone secretion. Thus, thereare interactions between brain, gland and a receptor with theinteractions proceeding in both directions. For example, TRH secretedfrom the brain hypothalamus stimulates pituitary TSH which acts tostimulate thyroid T₃ and T₄ which can act back on both pituitary andbrain in the form of both long (to brain) and short (to pituitary)feedback loops.

Henkin, R. I., Olfaction and Taste X I, (Kurihara, K., Suzuki, N.,Ogawa, H., Eds.), Springer Verlag, 1994, pp. 568-573, incorporatedherein by reference in its entirety, described the concept involvingsaliva and nasal mucus secretions related to taste and smell function(FIGS. 11 and 12). These results suggest that tastants and odorantsaffect brain function and vice versa. Since saliva and nasal mucus arethe critical factors in maintaining the taste and smell systems,respectively, it is understandable that substances in these fluids alsoaffect brain function and vice versa. Therefore, nasal administration ofsubstances can affect brain function and thereby affect variousphysiological and pathological problems. For example, nasaladministration of leptin (to control obesity), agouti-related protein(to increase appetite in anorexic patients), glucose, albumin, insulin(to treat diabetes), hormones (hormonal disorders), etc.

These effects may act through the large arteriovenous plexus of bloodvessels in the nose such that absorption of the substances may beenhanced by direct contact and absorption through these exposed vessels.FIGS. 11 and 12 reflect a feedback mechanism with effects acting fromnose to brain and from brain to nose, as in both a short and long loopfeedback system.

Treatment with Drug

Theophylline treatment restores smell function in some patients withhyposmia (loss of smell). Theophylline is a phosphodiesterase (PDE)inhibitor; it restores smell function through PDE inhibition therebyincreasing cAMP, a growth factor which stimulates maturation ofolfactory epithelial stem cells, cells whose functions are inhibitedamong patients with hyposmia. Theophylline may also restore smellfunction through other mechanisms. One such mechanism may operatethrough inhibition of excessive apoptosis, a normal process which, ifexcessively increased, can become pathological and impair cellularanatomy of the olfactory epithelium and cause hyposmia.

Table 16 illustrates detection and measurement of TRAIL in nasal mucusin patients with hyposmia before and after treatment with theophyllineat various doses. Data indicated that treatment with theophylline whichreturned smell function to normal in a dose-dependent manner wasassociated with a dose-dependent decrease in TRAIL. These data indicatethat treatment with a drug demonstrated a dose dependent decrease inTRAIL which indicates a decrease in the abnormal apoptotic processes.These data also indicate both a biochemical and functional improvementin smell function by treatment with theophylline. Without limiting thescope of the present invention, other drugs are also considered with inthe scope of the present invention for the treatment of variousdiseases. This is one of the example of the multiple examples of drugsto treat disease in which changes of various substances found in nasalmucus reflect biochemical normalization and functional improvement inthe disease process.

Table 33 in the examples illustrates NO in nasal mucus in patientstreated with theophylline in various doses before and after drugtreatment. NO levels in nasal mucus changed following the treatment ofpatients with smell loss. Results show treatment of patients with gradedincreasing doses of theophylline and measurement of both smell functionand NO in nasal mucus in patients with hyposmia. Results indicated thatprior to the treatment levels of NO in nasal mucus were lower than innormal subjects. After treatment with theophylline in graded doses therewere increases in nasal mucus NO associated with graded increases insmell function. These data demonstrate that treatment with drugs thatincrease smell function to or toward normal, returns smell function tonormal. These results demonstrate the measurements of various substancesin nasal mucus as an index of both human physiology and pathology ofvarious diseases. Its continual measurement during treatment of thedisorders helps in monitoring efficacy of therapy. The detection of NOin nasal mucus provides a non invasive method of diagnosing variousdiseases related to human physiology and pathology. The methods of thepresent invention include treatment of diseases by modulating theconcentrations of NO by use of drugs or agents. The method of treatmentis preferably by nasal administration.

Tables 36-38 illustrate detection and measurement of TNFα, TNFR 1 andTNFR 2 in nasal mucus of patients with graded loss of smell followingtreatment with theophylline. Results indicate that detection andmeasurements of TNFα, TNFR 1 and TNFR 2 in nasal mucus can be used as anindex of the disease process and of changes toward normal as the diseaseis successfully treated, in the present case with theophylline. Further,nasal mucus can be used as an index of disease, disease severity andefficacy of disease treatment. Without limiting the scope of the presentinvention, this approach is applicable to other substances in nasalmucus in relationship to other disease processes (e.g., cancer, stroke)and to follow-up of their treatment with any drug.

Treatment with Transcranial Magnetic Stimulation

Loss of taste and smell acuity (hypogeusia and hyposmia, respectively)with subsequent gustatory and olfactory distortions in the absence oforal or external olfactory stimuli [(phantageusia and phantosmia,respectively) labeled sensory distortions], are symptoms which may occurin some patients without other neurological or psychological disorders.

Transcranial magnetic stimulation (TCMS) use has been limited by lack ofobjective methods to measure efficacy of its application. One aspect ofthe invention includes method of treatment of patients with loss oftaste and/or smell (hypogeusia and/or hyposmia, respectively) withsubsequent gustatory and/or olfactory distortions (phantageusia and/orphantosmia, respectively) with repetitive TCMS (rTCMS) which improvedtheir sensory acuity and decreased their sensory distortions.

Increased CA VI secretion has been considered a marker for bothincreased taste and smell function. Thus, before and after rTCMS, CA VIactivity and other salivary proteins were measured in patients with bothsensory loss and presence of sensory distortions. Since CA VI is a zinccontaining glycose talloprotein, the salivary zinc and copperconcentrations were also measured to determine if changes in theseparameters correlated with changes in CA VI activity. The possibility ofthe changes in other salivary proteins was also investigated. Changes inerythrocyte CA I, II as well as concentrations of zinc and copper inboth erythrocytes and in blood plasma, were also measured.

Example 40 shows the study of ninety-three patients with hyposmia,hypogeusia, phantosmia and/or phantageusia before and after rTCMS.Measurements were made of activities of CA I, II in erythrocytes and ofCA VI, of concentrations of zinc and copper in parotid saliva, bloodserum, and erythrocytes and of appearance of proteins in saliva bySELDI-TOF mass spectrometry. Results showed that after rTCMS,significant increases occurred in CA I, II, CA VI, and in concentrationsof zinc and copper in blood plasma, erythrocytes and saliva. Salivaryproteins at m/z value of 21.5K with a repeating pattern at intervals of5K m/z were induced.

These results demonstrate the biochemical changes in specific enzymaticactivities and trace metal concentrations following rTCMS. These changesmay relate not only to several aspects of clinical abnormalities ofsensory function but also to other neurological disorders includingepilepsy, parkinsonism, alzheimer disease, head injury and motor neurondisease. Example 41 shows efficacy of treatment with rTCMS for patientswith these cognitive impairments such as hypogeusia, hyposmia,phantageusia, and phantosmia.

Other Example of Drugs

Drugs that may be used in the methods of treatment of the presentinvention may be selected from the following, viz. vaccination, alcoholabuse preparations, drugs used for Alzheimer's disease, anesthetics,acromegaly agents, analgesics, antiasthmatics, anticancer agents,anticoagulants and antithrombotic agents, anticonvulsants, antidiabeticsantiemetics, antiglaucoma, antihistamines, anti-infective agents,antiparkinsons, antiplatelet agents, antirheumatic agents,antispasmodics and anticholinergic agents, antitussives, carbonicanhydrase inhibitors, cardiovascular agents, cholinesterase inhibitors,treatment of CNS disorders, CNS; stimulants, contraceptives, cysticfibrosis management, dopamine receptor agonists, endometriosismanagement, erectile dysfunction therapy, fertility agents,gastrointestinal agents, immunomodulators and immunosuppressives, memoryenhancers, migraine preparations, muscle relaxants, nucleosideanalogues, osteoporosis management, parasympathomimetics,prostaglandins, psychotherapeutic agents, sedatives, hypnotics andtranquilizers, drugs used for slain ailments, steroids and hormones;Examples of alcohol abuse preparations are chlorazepate,chlordiazepoxide, diazepam, I disulfiram, hydroxyzine, naltrexone andtheir salts.

Examples of analgesics are acetaminophen, aspirin, bupivacain,boprenorphine, butorphanol, celecoxib, clofenadol, choline, clonidine,codeine, diflunisal, dihydrocodeine, dibydroergotamine, dihydromorphine,ethylmorphine, etodolac, eletriptan, eptazocine, ergotamine, fentanyl,fentoprofen, hyaluronic acid, hydrocodon, hydromorphon, hylan,ibuprofen, lindomethacin, ketorolac, lcetotifen, levomethadon,levallorphan, levorphanol, lidocaine, mefenamic acid, meloxicam,meperidine, metlladone, morphine, nabumetone, nalbuphin, nefopam,nalorphine, naloxone, naltrexone, naproxen, naratriptan, nefazodone,mormethadon, oxaprozin, oxycodone, oxymorphon, pentazocin, pethidine,phenpyramid, piritramid, piroxicam, propoxyphene, refecoxib,rizatriptan, salsalaketoprofen, sulindac, sumatriptan, tebacon, tilidin,tolmetin, tramadol, zolmitriptan and their salts.

Examples of antiasthmatics are ablukast, azelastine, bunaprolast,cinalukast, cromitrile, cromolyn, enofelast, isamoxole, ketotifen,levcromekalin, lodoxamide, montelukast, ontazolast, oxarbazole,oxatomide, piriprost potassium, pirolate, pobilukast edamine, quazolast,repirinast, ritolukast, sulukast, tetrazolastmeglumine, tiaramide,tibenelast, tomelukast, tranilast, verlukast, verofylline, szarirlukast.

Examples of anticancer agents are adriamycin, aldesleukin, allopurinol,altretamine, amifostine, anastrozole, asparaginase, betamethasone,bexarotene, bicalutamide, bleomycin, busulfan, capecitabine,carboplatin, cannustine, chlorambucil, cisplatin, cladarabine,conjugated estrogen, cortisone, cyclophosphamide, cylarabine,dacarbazine, daunorubicin, dactinomycin, denileukin, dexamethasone,discodermolide, docetaxel, doxorubicin, eloposidem, epirubicin, epoetin,epothilones, estramustine, esterified estrogen, ethinyl estradiol,etoposide, exemestane, flavopirdol, fluconazole, fludarabine,fluorouracil, flutamide, floxuridine, gemcitabine, gemtuzumab,goserelin, hexamethylmelamine, hydrocortisone, hydroxyurea, idarubicin,ifosfamide, interferon, irinotecan, lemiposide, letrozole, leuprolide,levamisole, levothyroxine, lomustine, mechlorethamine, melphalan,mercaptopurine mechlorethamine, megesterol, methotrexate,methylprednisolone, methyltestosterone, mithramycin, mitomycin,mitotane, mitoxantrone, mitozolomide, mutamycin, nilutamide, paclitaxel,pamidronate, pegaspargase, pentostatin, plicamycin, porfimer,prednisolone, procarbazine, rituximab, sargramostim, semustine,skeptozocin, tamoxifien, temozolomide, teniposide, testolactone,thioguanine, thiotepa, tomudex, topotecan, toremifene, trastumuzab,tretinoin, semustine, skeptozolocin, valrubicin, verteporfin,vinblastine, vincristine, vindesine, vinorelbine and their salts.

Examples of anticoagulants and antithrombic agents are warfarin,dalteparin, heparin, tinzaparin, enoxaparin, danaparoid, abciximab,alprostadil, altiplase, anagralide, aniskeplase, argatroban, ataprost,beraprost, camonagreel, cilostazol, clinprost, clopidogrel, cloricromen,dermatan, desirudin, domitroban, drotaverine, epoprostenol,eptifibatide, *adafiban, gabexate, iloprost, isbogrel, lamifiban,lamoteplase, le*adafiban, lepirudin, levosimendan, lexipafant,melagatran, nafagrel, nafamostsat, nizofenone, orbifiban, ozagrel,pamicogrel, parnaparin, quinobendan, reteplase, sarpogralate, satigrel,silteplase, simendan, ticlopidine, vapiprost, tirofiban, xemilofiban,Y20811 and their salts.

Examples of anticonvulsants are carbamazopine, clonazepam, clorazepine,diazepam, divalproex, ethosuximide, ethotion, felbamate, fosphenytoin,gabapentin, lamotrigine, levetiracetam, lorazepam, mephenytoin,mephobarbital, metharbital, methsuximide, oxcarbazopine, phenobarbital,phenytoin, primidone, tiagabine, topiramate, valproic acid, vigabatrin,zonisamide, and their salts. Examples of antidiabetic agents areacarbose, acetohexamide, carbutamide, chlorpropamide, epalrestat,glibornuride, gliclazide, glimepiride, glipizide, gliquidone,glisoxepid, glyburide, glyhexamide, metformin, miglitol, nateglinide,orlistat, phenbutamide, pioglitazone, repaglinide, rosiglitazone,tolazamide, tolbutamide, tolcyclamide, tolrestat, troglitazone,voglibose and their salts.

Examples of antiemetics are alprazolam benzquinamide, benztropine,betahistine, chlorpromazine, dexamethasone, difenidol, dimenhydrinate,diphenhydramine, dolasetron, domperidone, dronabinol, droperidol,granisetron, haloperidol, lorazepam, meclizine, methylprednisolone,metoclopramide, ondansetron, perphenazine, prochlorperazine,promethazine, scopolamine, tributine, triethylperazine, triflupromazine,trimethobenzamide, tropisetron and their salts.

Examples of antiglaucoma agents are alprenoxime, dapiprazole,dipivefrin, latanoprost, naboctate, pirnabine and their salts.

Examples of antihistamines are acrivastine, activastine, albuterol,azelastine, bitolterol, alimemazine, amlexanox, azelastine, benzydamine,brompheniramine, cetirizine, chlorpheniramine, cimetidine, clemastine,cycloheptazine, cyproheptadine, diclofenac, diphenhydramine, dotarizine,ephedrine, epinastine, epinephrine, ethyluorepinephrine, fenpoterol, 2sfexofenadine, flurbiprofen, hydroxyzine, ibuprofen, isoetharine,isoproterenol, ipratropium bromide, ketorolac, levocetirizine,loratidine, mequitazine, metaproterenol, phenylephrine, phenylprop anolamine, pirbuterol, promethazine, pseudo ephedrine, pyrilamine,salmeterol, terbutaline, tranilast, xanthine derivatives, xylometazolineand their salts.

Examples of anti-infective agents are abacavir, albendazole, amantadine,amphotericin, amikacin, aminosalicylic acid, amoxycillin, ampicillin,amprenavir, atovaquin, azithromycin, aztreonam, carbenicillin, cefaclor,cefadroxil, cefamandole, cefazolin, cefdinir, cefepime, cefexime,cefoperazone, cefotaxime, cefotitam, cefoperazone, cefoxitin,ceLpodoxine, cefprozil, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, cefuroxime, cephalexin, chloroquine, cidofovir, cilastatin,ciprofloxacin, clarithromycin, clavulinic acid, clindamycin,colistimethate, dalfopristine, dapsone, daunorubicin, delavirdin,demeclocycline, didanosine, doxycycline, doxorubicin, efavirenz,enoxacin, erythromycin, ethambutol, ethionamide, famsiclovir,fluconazole, flucytocin, foscarnet, fosfomycin, ganciclovir,gatifloxacin, griseofulvin, hydroxychloroquine, imipenem, indinavir,interferon, isoniazide, itraconazole, ivermectin, ketoconazole,lamivudine, levofloxacin, linezolide, lomefloxacin, lovacarbef,mebendazole, mefloquine, meropenem, methanamine, metronidazole,minocycline, moxefloxacin, nalidixic acid, nelfnavir, neomycin,nevirapine, nitrofurantoin, norfloxacin, ofloxacin, olseltamnivir,oxytetracycline, palivizumab, penicillins, perfloxacin, piperacillin,praziquantel, pyrazinamide, pyrimethamine, quinidine, quinupristine,retonavir, ribavirin, rifabutine, rifampicin, rimantadine, saquinavir,sparfloxacin, stavudine, streptomycin, sulfamethoxazole, teramycin,terbinafine, tetracycline, ticarcillin, thiabendazole, tobramycin,trimethoprim, trimetraxate, troleandomycin, trovafloxacin, valacyclovir,vancomycin, zalcitabine, zanamivir, zidovudine and their salts.

Examples of antiparkinsons are amantadine, adrogolide, altinicline,benztropine, biperiden, brasofensine, bromocriptine, budipine,cabergoline, dihydrexidine, entacapone, etilevodopa, idazoxan,iometopane, lazabemide, melevodopa, carbidopa/levodopa, mofegiline,moxiraprine, pergolide, pramipexole, quinelorane, rasagiline,ropinirole, seligiline, talipexole, tolcapone, trihexyphenidyl and theirsalts. Examples of antirheumatic agents are azathiprine, betamethasone,celecoxib, cyclosporin, diclofenac, hydroxychloroquine, indomethacin,infliximab, mercaptobutanedioic acid, methylprednisolone, naproxen,penicillamine, piroxicam, prednisolone, sulfasalazine and their salts.

Examples of platelet agents are abciximab, anagrelide, aspirin,cilostazol, clopidogrel, dipyridamole, epoprostenol, eptifbatide,ticlopidine, tinofban and their salts. Examples of antispasmodics andanticholinergic agents are aspirin, atropine, diclofenac, hyoscyamine,mesoprostol, methocarbamol, phenobarbital, scopolamine and their salts.

Examples of antitussives are acetaminophen, acrivastin, albuterol,benzonatate, beractant, brompheniramine, caffeine, calfactant,carbetapentane, chlorpheniramine, codeine, colfuscerin,dextromethorphan, dornase alpha, doxylamine, epinephrine, fexofenadine,guaiphenesin, iprakopium, levalbuterol, metaproterenol, montelukast,pentoxyphyline, phenylephrine, phenylpropanolamine, pirbuterol,poractant alpha, pseudoephedrine, pyrilamine, salbuterol, salmeterol,terbutaline, theophylline, zafirlukast, zileuton and their salts.Examples of carbonic anhydrase inhibitors are acetazolamide,dichlorphenamide, dorzolamide, methazolamide, sezolamide and theirsalts.

Examples of cardiovascular agents are abciximab, acebutolol, activase,adenosine, adrenaline, amidarone, amiloride, amlodipine, amyl nikate,atenolol, atorvastatin, benazepril, bepiridil, betaxalol, bisoprolol,candesartan, captopril, cartenolol, carvedilol, cerivastatin,chlorthalidone, chlorthiazole, clofibrate, clonidine, colestipol,colosevelam, digoxin, diltiazem, disopyramide, dobutamine, dofetilide,doxazosin, enalapril, epoprostenol, eprosartan, esmolol, ethacrynate,erythrityl, felodipine, fenoidapam, fosinopril, fleicainide,flurosemide, fluvastatin, gewhibrozil, hydrochlorthiazide,hydroflumethazine, ibutilide, indapamide, isosorbide, irbesartan,labetolol, lacidipine, lisinopril, losartan, lovastatin, mecamylamine,metoprolol, metaraminol, metazolone, methylchlorthiazide, methyldopa,metyrosine, mexiletine, midrodine, milrinone, moexipril, nadolol,niacin, nicardipine, nicorandil, nifedipine, nimodipine, nisoldipine,nikoglycerin, phenoxybenzamine, perindopril, polythiazide, pravastatin,prazosin, procainamide, propafenone, propranolol, quanfacine, quinapril,quinidine, ranipril, reteplase, simvastatin, sotalol, spironolactone,skeptokinase, telmisartan, terazosin, timolol, tocainamide, tors-emide,kandolapril, kiamterene, kapidil, valsartan and their salts.

Examples of cholinesterase inhibitors are donepezil, edrophonium,neostigmine, pyridostigmine, rivasti.gmine, tacrine and their salts.Examples of CNS stimulants are caffeine, doxapram, dexoamphetamine,donepezil, edrophonium, methamphetamine, methylphenidate, modafinil,neostigwine, pemoline, phentermine, pyridostigmine, rivastigwine, tacrinand their salts. Examples of cystic fibrosis management are dornasealpha, pancrelipase, tobramycin and their salts. Examples of dopaminereceptor agonists are amantadine, cabergoline, fenoldopam, pergolide,pramipexil, ropinirole and their salts. Examples of drugs used forendometriosis management are danazol, goserelin, leuprolide, nafarelin,norethindrone and their salts. Examples of drugs used for erectiledysfunction therapy are alprostadil, sildenafil, yohimbine andi theirsalts. I Examples of gastrointestinal agents are aldosetron, bisacodyl,bismuth subsalicylate, celecoxib, difoxin, dipheoxylate, docusate,famotidine, glycopyrrolate, infliximab, lansoprazole, loperamide,metaclopramide, nizatidine, omeprazole, pantoprazole, rabeprazole,ranitidine, simethicone, sucralfate, and their salts.

Examples of immunomodulators and immunosupressives are azathioprin,ceftizoxine, cyclosporin, daclizumab, glatiramer, immunoglobulin,interferon, leflunomide, levamisol, mycophenolate, mausomanab,phthalidomide, ribavirin, sirolimus and their salts. Examples of drugsused in Alzheimer's disease are donepezil, galanthamine, metrifonate,rivastigwine, tacrine, TAK-147 and their salts. Examples of drugs usedfor migraine preparations are acetaminophen, dihydroergotamine,divalproex, ergotamine, propranolol, risatriptan, sumatriptan,trimetrexate and their salts. Examples of muscle relaxants arealcuronium-chloride, azapropazon, atracurium, baclofen, carisoprodol,quinine derivatives, chloromezanon, chlorophenesincarbamate,chlorozoxazon, cyclobenzaprine, dantrolene, decamethoniumbromide,dimethyltubocurariniumchloride, doxacurium, fenyrami dol, gallamintriethio dide, guaiphenesin, hexafluoreniumbromide,hexacarbacholinbromide, memantin, mephenesin, meprobamate, metamisol,metaxalone, methocarbamol, mivacurium, orphenadrin, pancuronium,phenazon, phenprobamate, pip e curonium, rap acuronium, ro curonium, succinylcholine, soxamethoniumchloride, tetrazepam, tizanidine,tubocurarine chloride, tybamate, vecuronium and their salts.

Examples of nucleoside analogues are abacavir, acyclovir, didanosine,ganciclovir, gewcitabine, lamivudine, ribavirin, stavudine, zalcitabineand their salts. Examples of drugs used for osteoporosis management arealendronate, calcitonin, estradiol, estropipate, medroxyprogesterone,norethindrone, norgestimate, pamidronate, raloxifen, risdronate,zolendronate and their salts. Examples of parasympathomimetics arebethanechol, biperidine, edrophonium, glycopyrolate, i hyoscyamine,pilocarpine, tacrine, yohimbine and their salts. Examples ofprostaglandins are alprostadil, epoprostenol, misoprostol and theirsalts. Examples of psychotherapeutic agents are acetophenazine,alentemol, alpertine, alprazolam, amitriptyline, aripiprazole,azaperone, batelapine, befipiride, benperidol, benzindopyrine, bimithil,biriperone, brofoxine; bromperidol; bupropion, buspirone, butaclamol,butaperazine; carphenazine, carvotroline, cericlamine, chlorazepine,chlordiazepoxide, chlorpromazine; chlorprothixene, cinperene,cintriamide, citalopram, clomacran, clonazopam, clopenthixol,clopimozide, clopipazan, cloroperone, clothiapine, clothixamide,clozapine; cyclophenazine, dapiprazole, dapoxetine, desipramine,divalproex, dipyridamole, doxepin, droperidol, duloxetine, eltoprazine,eptipirone, etazolate, fenimide, fibanserin, flucindole, flumezapine,fluoxetine, fluphenazine, fluspiperone, fluspirilene, flutroline,fluvoxamine, gepione, gevotroline, halopemide, haloperidol, hydroxyzine,hydroxynortriptyline, iloperidone, imidoline, lamotrigine, loxapine,enperone, mazapertine, mephobarbital, meprobamate, mesoridazine,mesoridazine, milnacipran, mirtazapine, metiapine, milenperone,milipertine, molindone, nafadotride, naranol, nefazodone, neflumozide,ocaperidone, odapipam, olanzapine, oxethiazine, oxiperomide, pagoclone,paliperidone, paroxitene, penfluridol, pentiapine perphenazine,phenelzine, pimozide, pinoxepin, pipamperone, piperacetazine,pipotiazine, piquindone, pilindole, pivagabine, pramipexole,prochlorperazine, prochlorperazine, promazine, quetiapine, reboxetine,remoxipride, remoxipride, risperidone, rimcazole, robolzotan,selegiline, seperidol, sertraline, sertindole; seteptiline, setoperone,spiperone, sunipitron, tepiindole, thioridazine, thiothixene, tiapride,tioperidone, tiospione, topiramate, tranylcypromine, trifluoperazine,trifluperidol, triflupromazine, triflupromazine, kimipramine,venlafaxine, ziprasidone and their salts.

Examples of sedatives, hypnotics and tranquilisers are bromazepam,buspione, clazolam, clobazam, chlorazepate, diazepam, demoxepam,dexmedetomitine, diphenyhydramine, doxylamine, enciprazine, estrazolam,hydroxyzine, ketazolam, lorazatone, lorazepam, loxapine, medazepam,meperidine, methobarbital, midazolam, nabilone, nisobamate, oxazepam,pentobarbital, promethazine, propofol, triazolam, zalelplon, zolpidemand their salts. Examples of drugs used for treatment of skin ailmentsare acitretin, alclometasone, allitretinoin, betamethasone,calciprotrine, chlorhexidine, clobetasol, clocortolone, clotriamozole,collagenase, cyclosporin, desonide, difluorosone, doxepine,eflornithine, finasteride, fluocinolone, flurandrenolide, fluticasone,halobetasol, hydrochloroquine, hydroquinone, hydroxyzine, ketoconazole,mafenide, malathion, menobenzone, neostigmine, nystatin, podoflox,povidone, tazorotene, tretinoin and their salts.

Examples of steroids and hormones are alclometasone, betamethasone,calcitonin, cikorelix, clobetasol, clocortolone, cortisones, danazol,desmopressin, desonide, desogestrel, desoximetasone, dexamethasone,diflorasone, estradiol, estrogens, estropipate, ethynlestradiol, ifluocinolone, flurandrenolide, fluticasone, glucagon, gonadotropin,goserelin, halobetasol, hydrocortisone, leuprolide, levonorgestrel,levothyroxine, medroxyprogesterone, menotropins, methylprednisolone,methyltestosterone, mometasone, naferelin, norditropin, norethindrone,norgestrel, octreolide, oxandrolone, oxymetholone, polytropin,prednicarbate, prednisolone, progesterone, sermorelin, somatropin,stanozolol, testosterone, urofollitropin and their salts.

Example of agents that are susceptible to the gastric environment suchas proton pump inhibitors are pantoprazole, omeprazole, lansoprazole,esomeprazole, rabeprazole, pariprazole, leminoprazole, or an enantiomer,isomer, derivative, free base or salt thereof; lipid-lowering agentssuch as lovastatin, pravastatin, atorvastatin, simvastatin; agents thatare targeted to the intestine for local action such as 5-aminosalicylicacid, corticosteroids such as beclomethasone, budesonide, fluticasone,tixocortol useful in treating Crohn's disease and ulcerative colitis;agents that may be inactivated by the gastric contents such as enzymeslike pancreatin, antibiotics such as erythromycin; agents that causebleeding or irritation of the gastric mucosa such as aspirin, steroids,non-steroidal anti-inflammatory compounds like ibuprofen, naproxen,ketoprofen, fenoprofen, flurbiprofen, oxaprozin, diflunisal, diclofenac,indomethacin, tolmetin, sulindac, etodolac, acetaminophen, plateletinhibitors such as abciximab, intergrelin, dipyridamole; nucleosideanalogs such as didanosine, transfer factor preparations, hormones,insulin, and other agents that have decreased stability in the gastricenvironment, as well as agents that are requiredl for local action inthe latter part of the gastrointestinal Tact. The agents may be used astheir base or as their pharmaceutically acceptable salt or solvatethereof.

The treatment can be via oral administration, transmucosaladministration, buccal administration, nasal administration, inhalation,parental administration, intravenous, subcutaneous, intramuscular,sublingual, transdermal administration, and rectal administration. Nasaladministration is a preferred mode in the present invention. Nasaladministration may delay or obviate drug resistance that may occurthrough the other routes of administration, such as, oral or parentral.

Effective Dosages

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredient is contained in atherapeutically or prophylactically effective amount, i.e., in an amounteffective to achieve therapeutic or prophylactic benefit. Of course, theactual amount effective for a particular application will depend, interalia, on the condition being treated and the route of administration.Determination of an effective amount is well within the capabilities ofthose skilled in the art, especially in light of the disclosure herein.

Therapeutically effective amounts for use in humans can be determinedfrom animal models. For example, a dose for humans can be formulated toachieve circulating concentration that has been found to be effective inanimals. The amount administered can be the same amount administered totreat a particular disease or can be an amount lower than the amountadministered to treat that particular disease. Patient doses for oraladministration of the drug may range from about 1 μg-1 gm/day. Thedosage may be administered once per day or several or multiple times perday. The amount of the drug administered to practice methods of thepresent invention will of course, be dependent on the subject beingtreated, the severity of the affliction, the manner of administrationand the judgment of the prescribing physician. The dose used to practicethe invention can produce the desired therapeutic or prophylacticeffects, without producing serious side effects.

Routes of Administration

The methods of treatment in the invention include by way of exampleonly, oral administration, transmucosal administration, buccaladministration, nasal administration such as inhalation, parentaladministration, intravenous, subcutaneous, intramuscular, sublingual,transdermal administration, and rectal administration.

In some embodiments of the present invention, the method of treatment isby nasal administration or inhalation. Compositions for inhalation orinsufflation include solutions and suspensions in pharmaceuticallyacceptable, aqueous or organic solvents, or mixtures thereof, andpowders. The liquid or solid compositions may contain suitablepharmaceutically acceptable excipients as described supra. Thecompositions can be administered by the oral or nasal respiratory routefor local or systemic effect. Compositions in preferablypharmaceutically acceptable solvents may be nebulized by use of inertgases. Nebulized solutions may be inhaled directly from the nebulizingdevice or the nebulizing device may be attached to a face mask tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions may be administered, preferably orally ornasally, from devices that deliver the formulation in an appropriatemanner.

In some embodiments of the present invention, the method of treatment isby oral administration. Oral administration can be presented as discretedosage forms, such as capsules, cachets, or tablets, or liquids oraerosol sprays each containing a predetermined amount of an activeingredient as a powder or in granules, a solution, or a suspension in anaqueous or nonaqueous liquid, an oil-in-water emulsion, or awater-in-oil liquid emulsion. Such dosage forms can be prepared by anyof the methods of pharmacy, but all methods include the step of bringingthe active ingredient into association with the carrier, whichconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. For example, a tablet can be prepared by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets can be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as powder or granules, optionallymixed with an excipient such as, but not limited to, a binder, alubricant, an inert diluent, and/or a surface active or dispersingagent. Molded tablets can be made by molding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.

An active ingredient can be combined in an intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending on the form of preparation desired for administration. Inpreparing the compositions for an oral dosage form, any of the usualpharmaceutical media can be employed as carriers, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents, and the like in the case of oral liquid preparations(such as suspensions, solutions, and elixirs) or aerosols; or carrierssuch as starches, sugars, micro-crystalline cellulose, diluents,granulating agents, lubricants, binders, and disintegrating agents canbe used in the case of oral solid preparations, in some embodimentswithout employing the use of lactose. For example, suitable carriersinclude powders, capsules, and tablets, with the solid oralpreparations. If desired, tablets can be coated by standard aqueous ornonaqueous techniques.

Examples of suitable fillers for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

Disintegrants may be used in the method of treatment of the presentinvention to provide tablets that disintegrate when exposed to anaqueous environment. Disintegrants that can be used to formpharmaceutical compositions and dosage forms of the invention include,but are not limited to, agar-agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, other starches, clays, otheralgins, other celluloses, gums or mixtures thereof.

Lubricants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the pharmaceutical composition.

When aqueous suspensions and/or elixirs are desired for oraladministration, the essential active ingredient therein may be combinedwith various sweetening or flavoring agents, coloring matter or dyesand, if so desired, emulsifying and/or suspending agents, together withsuch diluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

The tablets can be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate canbe employed. Formulations for oral use can also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

Surfactant which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to,hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.That is, a mixture of hydrophilic surfactants may be employed, a mixtureof lipophilic surfactants may be employed, or a mixture of at least onehydrophilic surfactant and at least one lipophilic surfactant may beemployed. A solubilizer may also be added to increase the solubility ofthe hydrophilic drug and/or other components, such as surfactants, or tomaintain the composition as a stable or homogeneous solution ordispersion.

Mixtures of solubilizers may be used. Examples include, but not limitedto, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate,dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol,transcutol, propylene glycol, and dimethyl isosorbide. Particularlypreferred solubilizers include sorbitol, glycerol, triacetin, ethylalcohol, PEG-400, glycofurol and propylene glycol.

The compositions for the treatment can further include one or morepharmaceutically acceptable additives and excipients. Such additives andexcipients include, without limitation, detackifiers, anti-foamingagents, buffering agents, polymers, antioxidants, preservatives,chelating agents, viscomodulators, tonicifiers, flavorants, colorants,odorants, opacifiers, suspending agents, binders, fillers, plasticizers,lubricants, and mixtures thereof.

In addition, an acid or a base may be incorporated into the compositionto facilitate processing, to enhance stability, or for other reasons.Examples of pharmaceutically acceptable bases include amino acids, aminoacid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide,sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate,magnesium hydroxide, magnesium aluminum silicate, synthetic aluminumsilicate, synthetic hydrocalcite, magnesium aluminum hydroxide,diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine,triethylamine, triisopropanolamine, trimethylamine,tris(hydroxymethyl)aminomethane (TRIS) and the like. Suitable acids arepharmaceutically acceptable organic or inorganic acids. Examples ofsuitable inorganic acids include hydrochloric acid, hydrobromic acid,hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid,and the like. Examples of suitable organic acids include acetic acid,acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, aminoacids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonicacid, citric acid, fatty acids, formic acid, fumaric acid, gluconicacid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleicacid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid,propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid,succinic acid, tannic acid, tartaric acid, thioglycolic acid,toluenesulfonic acid, uric acid and the like.

The forms in which the compositions of the present invention may beincorporated for administration by injection include aqueous or oilsuspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, orpeanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueoussolution, and similar pharmaceutical vehicles.

Aqueous solutions in saline are also conventionally used for injection.Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and thelike (and suitable mixtures thereof), cyclodextrin derivatives, andvegetable oils may also be employed. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

The compositions for delivery can be formulated into preparations insolid, semi-solid, or liquid forms suitable for local or topicaladministration, such as gels, water soluble jellies, creams, lotions,suspensions, foams, powders, slurries, ointments, solutions, oils,pastes, suppositories, sprays, emulsions, saline solutions,dimethylsulfoxide (DMSO)-based solutions. In general, carriers withhigher densities are capable of providing an area with a prolongedexposure to the active ingredients. In contrast, a solution formulationmay provide more immediate exposure of the active ingredient to thechosen area.

In some embodiments of the present invention, the method of treatmentcan be transdermal. Transdermal patches may be used to providecontinuous or discontinuous infusion in controlled amounts, either withor without therapeutic agent. The construction and use of transdermalpatches for the delivery of pharmaceutical agents is well known in theart. See, e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Suchpatches may be constructed for continuous, pulsatile, or on demanddelivery of pharmaceutical agents.

Pharmaceutical compositions may also be prepared with one or morepharmaceutically acceptable excipients suitable for sublingual, buccal,rectal, intraosseous, intraocular, intranasal, epidural, or intraspinaladministration. Preparations for such pharmaceutical compositions arewell-known in the art. See, e.g., See, e.g., Anderson, Philip O.;Knoben, James E.; Troutman, William G, eds., Handbook of Clinical DrugData, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds.,Principles of Drug Action, Third Edition, Churchill Livingston, N.Y.,1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition,McGraw Hill, 20037 ybg; Goodman and Gilman, eds., The PharmacologicalBasis of Therapeutics, Tenth Edition, McGraw Hill, 2001; RemingtonsPharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000;Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (ThePharmaceutical Press, London, 1999); all of which are incorporated byreference herein in their entirety.

Kits

The invention also provides kits. As shown in FIG. 3, by way of exampleonly, the kit 300 may include a sterile nasal swab 301 for collection ofnasal secretions, an elongated storage and transport tube 302 forreceiving the swab wherein the tube can be glass or plastic and the tubemay have a replaceable end closure, and contain a sterile nutrientmedium for isolation of the nasal secretions, a sterile assay solution303 for addition to the transport tube, and a detector medium 304 forthe detection of a biological substance in the nasal secretion. The kitmay also include written instructions 305. In some embodiments, thetherapeutic agent can also be provided as separate compositions inseparate containers within the kit for the treatment. Suitable packagingand additional articles for use (e.g., measuring cup for liquidpreparations, foil wrapping to minimize exposure to air, and the like)are known in the art and may be included in the kit.

The following preparations and examples serve to illustrate theinvention. They should not be construed as narrowing it, or limiting itsscope.

EXAMPLES Example 1 PCR Analysis of a Specimen of Nasal Mucus

Specimen of nasal mucus from different subjects was collected andanalyzed using PCR. FIG. 4 depicts a polyacrylamide gel electrophoresisof samples as shown in Table 1. Lanes 1-7 in FIG. 4 reveal one majorband consistent with the presence of HLA. Lanes 8-14 reveal one majorband consistent with the presence of 13 globin. On the right are locatedmolecular weight markers of various KD.

TABLE 1 Results of PCR analysis of two samples of nasal mucus obtainedfrom normal subjects Area 3 (Units) Rot. Sample Rep. Sample Known Tm1Area 1 Tm2 Area2 Tm3 Ethidium Sample Pos. Name of . . . Type* Conc. (°C.) (Units) (° C.) (Units) (° C.) Bromide Comments 1 WB Pos U 84.0511.18 POS HLA Cont 2 R. Blk U 82.08 10.96 NEG HLA 3 Specimen # 1 U 83.919.602 POS HLA 4 Specimen # 2 U 84.74 10.86 POS HLA 5 Specimen # U 83.407.006 POS HLA 1 + Pos Cont 6 Specimen # U 84.63 8.658 POS HLA 1 + PosCont 7 R. Blk U 80.68 5.131 NEG HLA 8 WB Pos U 87.41 6.062 POS betaglobin Cont 9 R. Blk U 75.92 6.166 POS beta globin 10 Specimen # 1 U86.97 7.534 POS beta globin 11 Specimen # 2 U 76.91 5.492 86.91 8.009POS beta globin 12 Specimen # U 86.81 7.983 POS beta globin 1 + Pos Cont13 Specimen # U 86.54 7.451 POS beta globin 1 + Pos Cont 14 R. Blk U NEGbeta globin * P = Positive, U = Unknown, N = Negative, S = Standard, < >= De-Selected

Example 2 DNA Extraction Procedure from Body Fluids with QIAGEN Kit

All specimens and all reagents were equilibrated to room temperature. 20μl Qiagen Protease (or proteinase K) was pipetted into the bottom of a1.5 ml centrifuge tube. 200 μl of specimen (nasal mucus, plasma, saliva,urine and other biological fluids) was added to the centrifuge tube. Incase of specimens containing less than 1 μg of DNA or RNA, 5-10 μg ofcarrier DNA or RNA (20 μl of poly dA or 8 μl of poly [C]) was added. 200μl of AL buffer was added to the tube. Mixture was mixed bypulse-vortexing for 15 sec. Mixture was incubated at 56-60° C. for 15min. Mixture was vortexed and centrifuged briefly. 200 μl of ethanol(96-100%) was added to the tube. Mixture was vortexed and centrifugedbriefly. The mixture was carefully applied to a QIAamp spin column (in a2 ml collection tube) without wetting the rim. Caps of the columns wereclosed and the mixture was centrifuged for 1 min. The spin column wasplaced in another clean collection tube and the tube containing thefiltrate was discarded. 500 μl of buffer AW1 was added to the spincolumn, the lid was closed and the column was spun for 1 min. The columnwas placed in another clean collection tube and the tube containing thefiltrate was discarded. 500 μl of buffer AW2 was added to the spincolumn, the lid was closed and the column was spun at for 3 minutes; thecollection tube containing the filtrate was discarded. Since traceamounts of buffer AW2 inhibit PCR, complete removal of the buffer isdesirable. The column is then placed in a clean 1.5 ml centrifuge tubeand 50 μl of H₂O was added to it. It was incubated at room temperaturefor 15 minutes and centrifuged for 3 minutes. The resulting DNA solutioncan be stored at 4° C. for several months.

Example 3 LightCycler Data Analysis Report

TABLE 2 LightCycler Melting Analysis Report of Studies on each of twosamples of nasal mucus analyzed by PCR Program: denature Type: NoneCycles 1 Segment Temperature Hold Time Slope 2° Target Step Size StepDelay Acquisition Number Target (° C.) (sec) (° C.)/sec) Temp (° C.) (°C.) (Cycles) Mode 1 95 600 20 0 0 0 None Program: PCR Type:Qualification Cycles 45 Segment Temperature Hold Time Slope 2° TargetStep Size Step Delay Acquisition Number Target (° C.) (sec) (° C.)/sec)Temp (° C.) (° C.) (Cycles) Mode 1 95 10 20 0 0 0 None 2 58 15 20 0 0 0None 3 72 12 20 0 0 0 Single Program: melt Type: None Cycles 1 SegmentTemperature Hold Time Slope 2° Target Step Size Step Delay AcquisitionNumber Target (° C.) (sec) (° C.)/sec) Temp (° C.) (° C.) (Cycles) Mode1 95 0 20 0 0 0 None 2 50 60 20 0 0 0 None 3 95 0 0.2 0 0 0 ContinuousProgram: cool Type: None Cycles 1 Segment Temperature Hold Time Slope 2°Target Step Size Step Delay Acquisition Number Target (° C.) (sec) (°C.)/sec) Temp (° C.) (° C.) (Cycles) Mode 1 40 30 20 0 0 0 NoneFluorescence Settings 3.5 Melting Analysis Settings LED Power CALIBDisplay Mode Compatible Channel Setting F1/1 Color N/A Program Name MeltCompensation N/A Start Time 0:52:39.85 Stop Time 0:56:28.2 Car. MovementContinuous

FIG. 5 depicts LightCycler melting peak report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, temperature on abscissa. FIG. 6 depicts LightCycler dataanalysis report on results of PCR analysis of two samples of nasalmucus. Fluorescence is plotted on ordinate, cycle number on abscissa.FIG. 7 depicts LightCycler melting peak report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, temperature on abscissa. FIG. 8 depicts LightCycler dataanalysis report on results of PCR analysis of two samples of nasalmucus. Fluorescence is platted on ordinate, cycle number on abscissa.FIG. 9 depicts LightCycler data analysis report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, cycle number on abscissa.

Example 4 Analysis of Nasal Mucus Before and after Fasting

Table 3 depicts the results of the ELISA analysis of nasal mucuscollected from 49 subjects before (fasting) and after (non-fasting).FL/VOL is flow rate in ml/min, PROT is protein, LEP is leptin, LEP/PR isleptin/protein, AG is agouti related protein, AG/PR is agouti relatedprotein/protein, INS is insulin, INS/PR is insulin/protein, X is meanvalues of 49 subjects, and SD is standard deviation of results.

TABLE 3 Nasal mucus (nM) BEFORE AFTER BEFORE AFTER BEFORE AFTER BEFOREAFTER FL/VOL FL/VOL PROT PROT LEP LEP LEP/PR LEP/PR SUBJECT ml/minml/min mg/ml mg/ml pg/ml pg/ml ratio ratio  1 6.52 2.198 496 226  2 0.812.771 90  3 0.31 2.016 39  4 3.27 2.494  5 4.64 2.811 724 258  6 0.284.03 3.237 2.419 197 61  7 49.31 29.68 2.886 2.805 510 382 177 136  85.18 3.272  9 22.37 4.76 1.751 1.999 10 3 1.653 11 0.39 3.865 56 14 123.83 1.192 13 18.38 3.047 230 75 14 0.27 2365 15 1.37 2.834 16 8.362.252 34 15 17 9.9 2.644 28 11 18 29.13 21.63 3.859 3.093 45 51 12 16 192.93 1.584 18 11 20 1.32 2.114 21 5.84 2.068 22 5.94 1.63 2.16 2.673 10750 23 3 3.22 24 2.72 2.246 39 17 25 3.87 4.87 2.28 2.339 39 17 26 1.992.707 174 64 27 27.24 2.845 85 30 28 3 2.362 79 33 29 2.21 1.14 30 1.113.226 31 5.59 2.39 32 6.77 2.811 129 46 33 0.55 3.122 388 124 34 1.022.062 56 27 35 23.8 1.665 56 34 36 1.13 2.92 225 77 37 1.27 1.323 39 2938 3.33 3.537 45 13 39 6.39 1.901 287 151 40 1.27 1.356 135 100 41 1.222.379 183 77 42 3.35 2.72 2.609 2.892 73 107 28 37 43 10.23 1.123 124110 44 13.07 10.58 3.531 1.74 540 1265 153 727 45 4.15 2.552 22 9 462.76 3.83 3.012 3.859 107 124 36 32 47 3.34 3.37 143 42 48 10.42 3.233.335 2.419 104 56 31 23 49 1.09 2.016 X 7.21 6.77 2.55 2.46 276.0 182.270.5 95.9 SD 10.23 7.65 0.71 0.68 475.0 296.1 69.7 180.3 BEFORE AFTERBEFORE AFTER AG AG BEFORE AFTER INS INS BEFORE AFTER SUBJECT pg/ml pg/mlAG/PR AG/PR μIU/ml μIU/ml INS/PR INS/PR  1 1 0 22 10  2  3  4 7 3 10 4 5 7 2 7 2  6 1 8 0 3 5 16 2 7  7 13 7 5 2  8 7 2 31 9  9 2 1 15 5 9 310 7 4 23 14 11 6 2 8 2 12 1 1 6 5 13 4 1 10 3 14 15 5 2 1 0 16 17 18 911 2 4 4 12 1 4 19 2 1 19 12 20 8 4 11 5 21 3.5 2 15 7 22 3 2 1 1 11 165 6 23 7 2 9 3 24 7 3 8 4 25 21 8 9 3 43 48 19 21 26 5 2 9 3 27 7 2 9 328 7 3 43 18 29 9 8 8 7 30 8 2 31 10 31 7 3 74 31 32 33 1 0 95 30 34 353 2 13 8 36 8 3 19 7 37 38 39 6 3 40 6 4 27 20 41 0 0 25 11 42 2 9 1 3 412 2 4 43 3 3 2 2 44 2 1 51 24 14 14 45 16 6 46 6 10 2 3 14 15 5 4 47 72 10 3 48 4 3 1 1 4 6 1 2 49 6 3 X 5.5 6.1 2.3 2.6 18.6 18.0 7.5 8.1 SD4.0 3.0 1.9 1.2 20.6 11.8 7.5 6.4

Example 5 Analysis of Saliva Before and after Fasting

Table 4 depicts the results of the ELISA analysis of saliva collectedfrom 50 subjects before (fasting) and after (non-fasting). FL/VOL isflow rate in ml/min, PROT is protein, LEP is leptin, LEP/PR isleptin/protein, AG is agouti related protein, AG/PR is agouti relatedprotein/protein, INS is insulin, INS/PR is insulin/protein, X is meanvalues of 50 subjects, and SD is standard deviation of results.

TABLE 4 Saliva BEFORE AFTER BEFORE AFTER BEFORE AFTER FL/VOL FL/VOL PROTPROT LEP LEP BEFORE AFTER SUBJECT ml/min ml/min mg/ml mg/ml pg/ml pg/mlLEP/PR LEP/PR  1 0.456 2.483 10 4.03  2 0.61 2.546 7 2.75  3 0.673 2.6846 2.24  4 0.406 1.941  5 0.717 3.623 24 6.62  6 0.447 0.405 2.523 2.88617 6.74  7 0.572 1.119 3.946 3.491 2 4 0.51 1.15  8 0.405 3.473  9 0.3152.817 2.177 10 0.377 2.39 11 0.84 3.197 12 3.75 12 0.635 3.594 10 2.7813 0.721 3.306 8 2.42 14 0.908 3.433 5 1.46 15 0.908 3.277 16 0.5233.508 5 1.43 17 0.5 2.85 6 2.11 18 0.633 0.722 3.214 3.254 6 15 1.874.61 19 0.356 3.3 19 5.76 20 0.734 1.855 7 3.77 21 0.692 3.398 22 0.5843.646 23 1.211 0.705 3.145 3.295 11 3.50 24 0.601 2.483 25 0.634 1.37712 8.71 26 0.347 0.372 2.736 2.932 16 5.85 27 0.356 2.413 6 2.49 280.559 3.012 23 7.64 29 0.871 3.076 20 6.50 30 0.722 2.091 31 0.946 3.81932 0.355 2.989 33 0.496 3.669 11 3.00 34 0.569 3.456 16 4.63 35 0.343.358 12 3.57 36 0.766 2.033 15 7.38 37 0.969 3.024 6 1.98 38 0.8243.427 22 6.42 39 0.624 2.31 7 3.03 40 0.71 1.711 3 1.75 41 0.905 2.644 83.03 42 0.511 2.483 25 10.07 43 0.524 0.614 2.776 3.56 7 10 2.52 2.81 440.533 3.185 6 1.88 45 0.596 0.566 3.963 3.128 6 7 1.51 2.24 46 0.8062.068 7 3.38 47 1.041 0.908 3.295 2.748 11 17 3.34 6.19 48 0.714 2.863 00.00 49 0.524 0.631 3.4 2.638 10 19 2.94 7.20 50 0.841 2.65 X 0.64 0.642.93 2.96 11.4 9.8 4.1 3.3 SD 0.21 0.21 0.62 0.53 6.7 5.5 2.6 1.8 BEFOREAFTER BEFORE AFTER AG AG BEFORE AFTER INS INS BEFORE AFTER SUBJECT pg/mlpg/ml AG/PR AG/PR μIU/ml μIU/ml INS/PR INS/PR  1 15 6.04 17 6.85  2  3 4 6 3.09 20 10.30  5 8 2.21 21 5.80  6 6 8 2.38 2.77 15 12 5.95 4.16  79 9 2.28 2.58  8 2 0.58 10 2.88  9 8 3.67 6 4 2.13 1.84 10 7 2.93 2811.72 11 15 4.69 11 3.44 12 7 1.95 15 4.17 13 2 0.60 19 5.75 14 79 15 133.97 27 16 17 18 4 5 1.24 1.54 20 11 6.22 3.38 19 31 9.39 7 2.12 20 2513.48 21 10 2.94 10 2.94 22 46 12.62 43 11.79 23 6 8 1.91 2.43 13 2 4.130.61 24 8 3.22 20 8.05 25 7 5.08 27 19.61 26 10 7 3.65 2.39 9 2 3.290.68 27 5 2.07 9 3.73 28 8 2.66 17 5.64 29 7 2.28 12 3.90 30 10 4.78 4421.04 31 4 1.05 27 7.07 32 5 1.67 23 7.69 33 34 22 6.37 35 36 12 5.90 115.41 37 7 2.31 5 1.65 38 39 40 10 5.84 41 5 1.89 13 4.92 42 10 4.03 135.24 43 23 5 8.29 1.40 25 7 9.01 1.97 44 8 2.51 4 1.26 45 5 1.60 17 74.29 2.24 46 15 7.25 47 7 5 2.12 1.82 3 11 0.91 4.00 48 11 3.84 19 6.6449 3 14 0.88 5.31 21 3 6.18 1.14 50 7 2.64 X 10.7 6.7 3.7 2.5 18.4 11.16.2 3.7 SD 9.0 2.8 2.7 1.5 13.9 8.4 4.6 3.0

Example 6 Analysis of Plasma Before and after Fasting

Table 5 depicts the results of the ELISA analysis of plasma collected in20 subjects before (fasting) and after (non-fasting). PROT is protein inmg/dl, LEP is leptin, LEP/PR is leptin/protein, AG is agouti relatedprotein, AG/PR is agouti related protein/protein, INS is insulin, INS/PRis insulin/protein, X is mean values of 20 subjects, and SD is standarddeviation of result.

TABLE 5 Plasma BEFORE AFTER BEFORE AFTER BEFORE PROT PROT LEP LEP BEFOREAFTER AG SUBJECT mg/ml mg/ml pg/ml pg/ml LEP/PR LEP/PR pg/ml  1 7.8  27.2  3 7.7  4 8.6  5 7.9 10661 1349 30  6 6.5 5345  7 6.9 51  8 7.6 38 9 7.9 34 10 7.4 11 7.1 53 12 6.2 6.2 1793 1714 289 276 65 13 7.2 5075705 25 14 7.2 15 6.6 16 7.2 16860 2342 17 7.9 16860 2134 18 7.3 158262168 19 7.3 20 6.6 X 7 7 5843 11321 781 1730 42 SD 1 1 4484 7240 534 97314 AFTER BEFORE AFTER AG BEFORE AFTER INS INS BEFORE AFTER SUBJECT pg/mlAG/PR AG/PR μIU/ml μIU/ml INS/PR INS/PR  1 35 4 55 7  2 38 5  3 37 5 365  4 61 7 46 5  5 4 3 0  6 51 8 1 0  7 7 17 2  8 5  9 4 41 5 10 28 4 6 111 7 12 10 95 60 15 10 13 3 4 1 14 70 10 24 3 15 34 5 16 55 8 44 6 17 334 1 0 18 37 5 7 1 19 37 5 18 2 20 58 9 X 45 6 6 32 28 5 4 SD 14 3 2 3821 6 3

Example 7 Analysis of Insulin Concentration in Nasal Mucus, Plasma, andSaliva

Specimens of nasal mucus from different subjects were collected andanalyzed using ELISA. Table 6 depicts detection and measurement of humaninsulin in nasal mucus as compared to insulin in blood plasma and salivaunder several physiological and pathological processes. In controlsubjects, in the fasting state, insulin concentrations were similar ineach biological fluid measured. In the non-fasting state, the nasalmucus concentrations were significantly lower than in plasma or saliva.In obese subjects and in diabetics, in the fasting state, insulinconcentrations were similar in plasma and nasal mucus but slightlyelevated in saliva. However, in the non-fasting state, insulinconcentrations increased in plasma and saliva in response to increasedcarbohydrate intake but in nasal mucus, insulin levels decreased.

TABLE 6 Insulin in plasma, saliva, and nasal mucus PLASMA NASAL AGEWEIGHT GLUCOSE PLASMA SALIVA MUCUS SUBJECTS (years) (lbs) mg/ml μIU/mlμIU/ml μIU/ml CONTROLS (56) 56 ± 2 174 ± 8  FASTING 88 ± 1 17.1 ± 3.718.9 ± 2.3 19.0 ± 2.2 NON-FASTING 174 ± 8  29.4 ± 4.4 22.6 ± 2.6  7.5 ±0.9^(c) SUBJECTS (11) OBESE 51 ± 6 259 ± 26 FASTING 110 ± 24 20.0 ±6.2^(†) 34.1 ± 7.9 18.4 ± 6.5 NON-FASTING 259 ± 26 38.2 ± 11.7 36.3 ±6.3  7.5 ± 2.6^(d b a) DIABETES 51 ± 6 243 ± 52 FASTING 142 ± 37 22.7 ±10.2 36.8 ± 9.0 21.0 ± 9.3 NON-FASTING 243 ± 42 39.5 ± 19.0 38.2 ± 9.6 7.6 ± 2.3^(b) ( ) Subject number ^(†)MEAN ± SEM ^(a)p < 0.01 withrespect to non-fasting saliva ^(b)p < 0.005 with respect to fastingsaliva ^(c)p < 0.001 with respect to fasting nasal mucus and non-fastingplasma, saliva ^(d)p < 0.05 with respect to fasting plasma, non-fastingplasma ^(e)p < 0.02 with respect to fasting plasma

Example 8 Analysis of Insulin Receptor Concentration in Nasal Mucus,Plasma, and Saliva

Table 7 depicts the detection and measurement of human insulin receptorin nasal mucus as compared to the insulin receptor in plasma and salivaunder several physiological and pathological conditions. In controlsubjects in the fasting state, insulin receptor concentrations measuredwere similar in plasma, saliva as well as nasal mucus. However, in thenon-fasting state, where there was little change in receptorconcentrations in plasma or saliva, there was a significant decrease innasal mucus receptor concentration. In obese subjects and in diabetics,in the fasting state, insulin receptor concentrations decreased in eachbiological fluid measured. However, in the non-fasting state, there wereno further decreases in receptor concentrations in plasma or in salivabut there was a decrease in receptor concentration in nasal mucusassociated with an increase in carbohydrate intake.

TABLE 7 Insulin receptor concentration in plasma, saliva, and nasalmucus PLASMA NASAL AGE WEIGHT GLUCOSE PLASMA SALIVA MUCUS SUBJECTS (56)(years) (lbs) mg/ml mg/ml mg/ml mg/ml CONTROLS (56) 56 ± 2^(†) 174 ± 8  84 ± 1^($) FASTING  88 ± 6 8.7 ± 1.8^(†) 7.6 ± 0.7 8.5 ± 1.7NON-FASTING 174 ± 8 7.5 ± 1.4 7.6 ± 0.6 3.2 ± 0.6^(a) SUBJECTS (11)OBESE 51 ± 6^(‡) 259 ± 26 FASTING 110 + 24$ 2.9 ± 0.9^(† b) 6.0 ± 1.24.8 ± 0.6^(d) NON-FASTING 259 + 26 3.0 ± 0.3^(b) 5.2 ± 0.8^(c) 2.1 ± 0.5THIN 122 ± 6  FASTING 8.4 ± 2.7 NON-FASTING 9.4 ± 4.6 DIABETES (4) 243 ±52 FASTING 142 ± 37 4.0 ± 1.7^(b) 4.0 ± 0.6^(c) 4.7 ± 1.9 NON-FASTING243 ± 42 3.4 ± 0.6^(a) 4.6 ± 1.9 2.3 ± 1.6 ^(†)MEAN ± SEM ^(‡)years^($)mg/dl ^(a)p < 0.01 with respect to nasal mucus fasting, plasma(fasting, non-fasting), saliva (fasting, non-fasting) ^(b)p < 0.005 withrespect to controls ^(c)p < 0.001 with respect to controls ^(d)p < 0.05with respect to controls

These results in Tables 6 and 7 indicate that the characteristics ofnasal mucus reflect physiological and pathological conditions. Thedetection of the presence of insulin or insulin receptors in nasal mucusmay offer an alternative method for diagnosis of diabetes, otherdisorders of carbohydrate metabolism and physiological measurements ofinsulin and insulin receptors. Its ease of measurement using anon-invasive technique may be preferable to invasive techniques such asvenipucture. Its presence in nasal mucus may also offer a view intoother aspects of both human physiology and pathology. In the non-fastingstate insulin receptor concentration is down regulated to some extentunder some conditions but is uniformly regulated in nasal mucusindicating that, its concentration in nasal mucus may indicatephysiological phenomenon such as, appetite and brain, and the signalcharacteristics reflecting base human physiology and pathology. Itspresence may influence human immune and autoimmune responses.

Example 9 Pearson-Product-Moment Correlations of Insulin with PlasmaGlucose and Insulin with Weight

Table 8 depicts Pearson-product-moment correlations of insulin withplasma glucose and insulin with weight. There was little positivecorrelation among controls in insulin in plasma or insulin in saliva, ineither the fasting or non-fasting state. However, in nasal mucus thiscorrelation was negative, indicating a down regulated direction. Thissignal in nasal mucus may reflect a control mechanism of appetite. Thus,in nasal mucus there may be substances which reflect both physiologicalparameters common or uncommon to blood or saliva which provides methodsto diagnose body physiological and pathological events.

TABLE 8 Correlation (pearson product moment (r)) between insulin andindependent variable (N = 60) INSULIN CONCENTRATION NASAL PLASMA (r)SALIVA (r) MUCUS (r) NON- NON- NON- FAST- FAST- FAST- FAST- FAST- FAST-ING ING ING ING ING ING VARIABLE (CONTROLS) PLASMA 0.16 0.08 0.25 0.26−0.20 −0.14 GLUCOSE (mg/dl) WEIGHT(lbs) 0.04 0.24 0.26 0.38 −0.14 −0.18VARIABLE (OBESE) PLASMA 0.87 −0.01   0.33 0.39 −0.30 −0.37 GLUCOSE(mg/dl) WEIGHT(lbs) 0.62 0.72 0.08 0.65 −0.44 −0.55 VARIABLE (DIABETES)PLASMA  1.00^(a)  1.00^(a) 0.65 0.59 −0.26 −0.64 GLUCOSE (mg/dl)WEIGHT(lbs) 1.00  0.99^(a) 0.69 0.69 −0.28 −0.66 ^(a)p < 0.01

Example 10 Pearson-Product-Moment Correlations of Insulin Receptor withPlasma Glucose and Insulin Receptor with Weight

Table 9 depicts relationships between insulin receptor concentrationwith plasma glucose and weight. There was little positive correlationamong controls amongst insulin receptors in plasma or insulin receptorsin saliva, in either the fasting or non-fasting state. However, in nasalmucus this correlation was negative, indicating a down regulateddirection. This signal in nasal mucus may reflect a control mechanism ofappetite. Thus, in nasal mucus there may be substances which reflectboth physiological parameters common or uncommon to blood or salivawhich may provide methods to diagnose body physiological andpathological events.

TABLE 9 Correlation (pearson product moment (r)) between insulinreceptor concentration and independent variable (n = 60) INSULINRECEPTOR CONCENTRATION NASAL PLASMA (r) SALIVA (r) MUCUS (r) NON- NON-NON- FAST- FAST- FAST- FAST- FAST- FAST- ING ING ING ING ING INGVARIABLE (CONTROLS) PLASMA   0.20   0.12 −0.24 −0.14 0.03   0.11 GLUCOSE(mg/dl) WEIGHT(lbs) −0.11 −0.07 −0.08 −0.19 −0.21   −0.17 VARIABLE(OBESE) PLASMA   0.70   0.20 −0.51 −0.08 0.41   0.19 GLUCOSE (mg/dl)WEIGHT(lbs) −0.18 −0.33   0.09 −0.16 0.24 −0.09 VARIABLE (DIABETES)PLASMA −0.57 −0.43 −0.78   0.25 0.15 −0.22 GLUCOSE (mg/dl) WEIGHT(lbs)−0.60 −0.52   0.56 −0.34 0.18 −0.19

Example 11 Analysis of Caspase 3 in Nasal Mucus and Saliva

Caspase 3 is one of the apoptotic substances involved in the apoptoticprocess. Table 10 illustrates a comparison between the detection andmeasurement of caspase 3 in nasal mucus and in saliva in 18 subjects.The presence of caspase in nasal mucus is about 13% of that in salivaand reflects the magnitude of the apoptotic process. The presence ofcaspase in nasal mucus indicates the activity of cellular death in bothphysiological and pathological processes.

TABLE 10 Caspase 3 in human saliva and nasal mucus SALIVA NASAL MUCUSCASPASE 3 PROTEIN CASPASE 3 CASPASE 3 PROTEIN CASPASE 3 Subjects μg/mlmg/dl PROTEIN μg/ml mg/dl PROTEIN 18 2.87 ± 0.70^(†) 2.7 ± 0.2 2.88 ±0.28 0.38 ± 0.13 2.2 ± 0.2 0.38 ± 0.17 ^(†)MEAN ± SEM

Example 12 Analysis of TNFα in Nasal Mucus and Saliva

Table 11 illustrates detection and measurement of TNFα in nasal mucusand saliva in 75 subjects. Results indicate that TNFα in nasal mucus isabout 30 times higher than in saliva. Elevated levels of this substancein nasal mucus in diverse disease processes makes their diagnosispossible on a clinical basis since obtaining cellular diagnosis throughtissue biopsy can not only be invasive but also difficult and at timesdangerous. The concentration of TNFα in nasal mucus can be reflective ofunderlying disease processes and is easier to obtain. These results maymake the use of this fluid an important method of diagnosis for thesepathological processes which cannot be as conveniently made in plasma.These data suggest that various cancers can be diagnosed by measurementsof TNFα in nasal mucus and their treatment can be monitored by followingits concentration in nasal mucus. Since levels of TNFα may also reflectthe inflammatory aspects of disease processes inducing it, use of antiTNFα drugs through nasal administration may reflect a method of treatingthese various disease processes. Concentrations of TNFα in nasal mucusin patients with smell loss can be greater than for example, 5000 timesthat found in normal subjects thereby reflecting its function as a“death protein” indicator of excessive apoptosis as in its role incancer.

TABLE 11 TNFα in human saliva and nasal mucus SALIVA NASAL MUCUS TNFαPROTEIN TNFα TNFα PROTEIN TNFα Subjects μg/ml mg/dl PROTEIN μg/ml mg/dlPROTEIN 75 0.43 ± 0.03^(†) 3.1 ± 0.1 0.15 ± 0.01 12.8 ± 1.6^(a) 2.4 ±0.09^(a) 5.5 ± 1.3^(a) ^(†)MEAN ± SEM With respect to saliva ^(a)p <0.001

Example 13 Analysis of TNFR I in Nasal Mucus and Saliva

Table 12 illustrates detection and measurement of TNFR I in 47 subjects.Results indicate that TNFR I in nasal mucus is about 16 times itsconcentration in saliva and its concentration is significantly increasedover that found in plasma, red blood cells, or urine. The results showthat TNFR I present in nasal mucus can reflect activity of manyinflammatory, oncological and other pathological processes, includingtaste and smell dysfunction. Thus the methods of the present inventioncan be used to detect and establish clinical diagnoses of excessiveapoptosis and can be used as a treatment modality in inhibitingpathological apoptosis.

TABLE 12 TNF receptor I in human saliva and nasal mucus SALIVA NASALMUCUS TNFR I PROTEIN TNFR I TNFR I PROTEIN TNFR I Subjects μg/ml mg/dlPROTEIN μg/ml mg/dl PROTEIN 47 110 ± 13^(†) 3.1 ± 0.1 38 ± 5 1753 ±357^(a) 2.2 ± 0.1^(a) 837 ± 161^(a) ^(†)MEAN ± SEM With respect tosaliva ^(a)p < 0.001

Example 14 Analysis of TNFR II in Nasal Mucus and Saliva

Table 13 illustrates detection and measurement of TNFR II in 47subjects. Results indicate that TNFR II in nasal mucus is about 24 timesits concentration in saliva and its concentration in nasal mucus issignificantly higher than found in plasma, red blood cells, or urine.The results reflect that TNFR II present in nasal mucus provides a noninvasive method of diagnosing various pathological processes. Thus themethods of the present invention can be used to detect and establishclinical diagnoses of excessive apoptosis and can be used as a treatmentmodality in inhibiting pathological apoptosis.

TABLE 13 TNF receptor II in human saliva and nasal mucus SALIVA NASALMUCUS TNFR II PROTEIN TNFR II TNFR II PROTEIN TNFR II Subjects μg/mlmg/dl PROTEIN μg/ml mg/dl PROTEIN 47 48 ± 2^(†) 3.1 ± 0.1 17 ± 0.9 1126± 217^(a) 2.2 ± 0.1^(a) 578 ± 128^(a) ^(†)MEAN ± SEM With respect tosaliva ^(a)p < 0.001

Example 15 Analysis of TRAIL in Nasal Mucus and Saliva

Tables 14 and 15 illustrate detection and measurement of TRAIL in salivaand nasal mucus in normal subjects and in patients with smell loss. InTable 14, results indicate that TRAIL in nasal mucus is about 5 timeshigher than in saliva and both are significantly higher than in blood,red blood cells, or urine. TRAIL in nasal mucus in some patients withsmell and taste loss varies from 500-10,000 times higher than in normalsubjects and it may be elevated in nasal mucus in patients withinflammatory and oncological disease processes. Mean levels of increasedTRAIL in nasal mucus reveal levels significantly greater than in otherfluids. The results provide a non-invasive method for detecting TRAIL innasal mucus.

TABLE 14 Trail in saliva and nasal mucus in normal subjects and inpatients with smell loss SALIVA NASAL MUCUS TRAIL PROTEIN TRAIL TRAILPROTEIN TRAIL SUBJECTS (Number) μg/ml mg/dl PROTEIN μg/ml mg/dl PROTEINNORMALS(17) 336 ± 48^(†) 2.7 ± 0.1 122 ± 14 1639 ± 89   2.2 ± 0.01 770 ±35  PATIENTS(10) 335 ± 31   2.7 ± 0.2 141 ± 20 2584 ± 430 1.4 ± 0.2 4753± 1400 ^(†)MEAN ± SEM

TABLE 15 Trail in saliva and nasal mucus in normal subjects and inpatients with Hyposmia SALIVA FLOW TRAIL NASAL MUCUS SUBJECTS PROTEINTRAIL RATE FLOW PROTEIN TRAIL TRAIL (Number) mg/dl μg/ml ml/sec RATEmg/dl μg/ml PROTEIN NORMALS(28) 2.29 ± 0.10^(†) 123 ± 6   0.80 ± 0.03343 ± 13  2.65 ± 0.12 1990 ± 119 7.56 ± 28   PATIENTS(65) 2.96 ± 0.09  330 ± 25^(a) 0.63 ± 0.03 582 ± 55^(a) 1.97 ± 0.11 4121 ± 54^(a ) 3095 ±591^(a) ( )Subject number ^(†)Mean ± SEM ^(a)p < 0.001 with respect tonormal

Example 16 Analysis of TRAIL in Nasal Mucus in Patients with HyposmiaBefore and after Treatment with Theophylline

Table 16 illustrates detection and measurement of TRAIL in nasal mucusin patients with hyposmia before and after treatment with theophyllineat various doses. Data indicate that treatment with theophylline whichreturned smell function to normal in a dose-dependent manner wasassociated with a dose-dependent decrease in TRAIL, which indicates adecrease in the abnormal apoptotic processes. These data indicate both abiochemical and functional improvement in smell function by treatmentwith theophylline.

TABLE 16 Nasal mucus Trail in patients with hyposmia before and aftertreatment with theophylline at various doses PRE- THEOPHYLLINE TREATMENTTREATMENT 200 mg 400 mg 600 mg SUBJECTS TRAIL PROTEIN TRAIL PROTEINTRAIL PROTEIN TRAIL PROTEIN (Number) μg/ml mg/dl μg/ml mg/dl μg/ml mg/dlμg/ml mg/dl PATIENTS 2584 1.36 855 1.29 791 2.06 247 2.00 (5) NORMALS 335 (9) ^(†) MEAN ± SEM

Example 17 Analysis of IL2 in Saliva and Nasal Mucus

Table 17 illustrates levels of IL2 in human nasal mucus and saliva. IL2was not present in nasal mucus although it was found in plasma.

TABLE 17 IL 2 in human saliva and nasal mucus SUB- SALIVA NASAL MUCUSJECTS IL 2 PROTEIN IL 2 IL 2 PROTEIN IL 2 (Number) μg/ml mg/dl PROTEINμg/ml mg/dl PROTEIN (10) 0 3.1 ± 0.2^(†) 0 0 2.2 ± 0.1 0 ^(†)MEAN ± SEM

Example 18 Analysis of IL3 in Saliva and Nasal Mucus

Table 18 illustrates measured IL 3 in both human saliva and nasal mucus.Levels of IL 3 in nasal mucus were found to be about ½ the concentrationin saliva but both levels were higher than that found in plasma, redblood cells, or urine. IL 3 present in nasal mucus provides a noninvasive method of diagnosing various IL3 related diseases.

TABLE 18 IL 3 in human saliva and nasal mucus SALIVA NASAL MUCUS PRO- IL3 PRO- IL 3 SUBJECTS IL 3 TEIN PRO- IL 3 TEIN PRO- (Number) μg/ml mg/dlTEIN μg/ml mg/dl TEIN (17) 140 ± 32^(†) 3.1 ± 0.2 48 ± 15 63 ± 24 2.2 ±0.1 43 ± 12 ^(†)MEAN ± SEM

Example 19 Analysis of Endostatin in Human Plasma, Urine, Saliva andNasal Mucus

Table 19 illustrates detection and measurement of endostatin in plasma,urine, saliva and nasal mucus in 15 subjects. Endostatin levels in nasalmucus were 5 times higher than in saliva and about 7% of that found inplasma. On the basis of the endostatin/protein ratio, levels in nasalmucus are about 14% of that found in plasma. Presence of this 20 KDprotein in nasal mucus illustrates a non-invasive method of detection ofendostatin in nasal mucus and its use in diagnosing various endostatinrelated diseases. It may also be indicative of its synthesis in nasalmucus.

TABLE 19 Endostatin in human plasma, urine, saliva and nasal mucusBIOLOGICAL ENDOSTATIN PROTEIN ENDOSTATIN FLUIDS (15) μg/ml mg/dl PROTEINPLASMA 94 ± 10^(†)  6.9 ± 0.10 15 ± 1   URINE  0.5 ± 0.04^(a) — — SALIVA1.3 ± 0.3^(a) 2.6 ± 0.2 0.59 ± 0.04^(a) NASAL MUCUS 6.6 ± 1.3^(a) 3.0 ±0.2  2.0 ± 0.43^(a) ( ) Subject number ^(†)MEAN ± SEM ^(a)p < 0.001 withrespect to plasma

Example 20 Analysis of Erythropoetin (EPO) in Plasma, Urine, Saliva andNasal Mucus

Table 20 illustrates detection and measurement of EPO in plasma, urine,saliva and nasal mucus. EPO was not found in urine or saliva. The levelof EPO in nasal mucus was found to be between 1.1 and 4.5 times higherthan in plasma. Presence of EPO in nasal mucus illustrates anon-invasive method of detection of EPO in nasal mucus and its use indiagnosing various EPO related diseases.

TABLE 20 EPO in plasma, urine, saliva and nasal mucus ERYTHRO-BIOLOGICAL ERYTHROPOETIN PROTEIN POETIN FLUIDS (27) μIU/ml mg/dl PROTEINPLASMA 13 ± 2^(†) 7.2 ± 0.1   2 ± 0.3 URINE 0 0 0 SALIVA 0 0 0 NASALMUCUS 15 ± 5  2.9 ± 0.1^(a) 9 ± 2^(b) ( ) Subject number ^(†)MEAN ± SEM^(a)p < 0.001 with respect to plasma ^(b)p < 0.005 with respect toplasma

Example 21 Analysis of Bone Morphogenic Protein (BMP) in Plasma, Urine,Saliva and Nasal Mucus

Table 21 illustrates detection and measurement of BMP I in plasma,urine, saliva and nasal mucus in 20 subjects. BMP I was found in plasmabut not in urine, saliva or nasal mucus.

TABLE 21 BMP in plasma, urine, saliva and nasal mucus BMP PROTEIN BMPBIOLOGICAL FLUIDS (20) μg/ml mg/dl PROTEIN PLASMA 30 ± 6^(†) 3.6 ± 0.5 0URINE 0 0 0 SALIVA 0 0 0 NASAL MUCUS 0 0 0 ( ) Subject number ^(†)MEAN ±SEM

Example 22 Analysis of Brain Derived Neurotrophic Factor (BDNF) in HumanPlasma, Urine, Saliva and Nasal Mucus

Table 22 illustrates detection and measurement of BDNF in plasma, urine,saliva and nasal mucus in 20 subjects. BDNF was found in plasma andnasal mucus but not in urine or saliva. Levels of BDNF in plasma werehigher than in nasal mucus. The results indicate that nasal mucus may bea repository of the family of nerve growth factors and the concentrationof BDNF as shown in Table 22, may help understand both the physiologyand pathology of neurotrophic factors related to growth and homeostasisof cells in the nasal cavity as well as reporting on the presence ofthese factors in the systemic circulation.

TABLE 22 BDNF in human plasma, urine, saliva and nasal mucus BDNFPROTEIN BDNF BIOLOGICAL FLUIDS (20) μg/ml mg/dl PROTEIN PLASMA 3391 ±530  7.1 ± 0.01 447 ± 74 URINE 0 — SALIVA 0 2.7 ± 0.2 NASAL MUCUS 11 ± 73.2 ± 0.3  8 ± 6 ( ) Subject number ^(†)MEAN ± SEM

Example 23 Analysis of Ciliary Neurotrophic Factor (CNTF) in HumanPlasma, Urine, Saliva and Nasal Mucus

Table 23 illustrates detection and measurement of CNTF in plasma, urine,saliva and nasal mucus in 19 subjects. Levels of CNTF in plasma andnasal mucus were found to be similar but lower in saliva. These resultsindicate that measurement of CNTF in nasal mucus may be used as an indexfor the levels of CNTF in plasma. The results provide a non-invasivemethod for the detection of CNTF in nasal mucus. The detection of CNTFin nasal mucus provides a non invasive method of diagnosing various CNTFrelated diseases.

TABLE 23 CNTF in human plasma, urine, saliva and nasal mucus CNTFPROTEIN CNTF BIOLOGICAL FLUIDS (19) μg/ml mg/dl PROTEIN PLASMA 0.004 ±0.001 3.1 ± 0.1 0 URINE 0 SALIVA 0.001 ± 0.001 3.0 ± 0.1 0 NASAL MUCUS0.003 ± 0.001 2.2 ± 0.1 0 ( ) Subject number

Example 24 Analysis of Granulocyte Macrophage Growth Factor (GM-CSF) inHuman Plasma, Urine, Saliva and Nasal Mucus

Table 24 illustrates detection and measurement of GM-CSF in plasma,urine, saliva and nasal mucus in 16 subjects. Levels in nasal mucus werefound to be over 6 times that found in plasma. The results provide anon-invasive method for the detection of GM-CSF in nasal mucus. Theresults also indicate nasal mucus to be a source of GM-CSF. Thedetection GM-CSF present in nasal mucus provides a non invasive methodof diagnosing various GM-CSF related diseases.

TABLE 24 GM-CSF in human plasma, urine, saliva and nasal mucusBIOLOGICAL GM-CSF PROTEIN GM-CSF FLUIDS (16) μg/dl mg/dl PROTEIN PLASMA0.42 ± 0.31^(†) 7.2 ± 0.1 0.036 ± 0.034 URINE 0 0 SALIVA 0 0 NASAL MUCUS2.55 ± 0.8   2.9 ± 0.1  0.58 ± 0.25^(a) ( ) Subject number ^(†)MEAN ±SEM ^(a)p < 0.001 with respect to plasma

Example 25 Analysis of Hepatocyte Growth Factor (HGF) in Human Plasma,Urine, Saliva, and Nasal Mucus

Table 25 illustrates detection and measurement of HGF in plasma, urine,saliva and nasal mucus in 17 subjects. Concentrations of HGF in nasalmucus were found to be higher than that found in either plasma or urine.These results suggest that HGF may be synthesized in the serous glandsof the nose for a specific mechanism involved with nasal homeostasis aswell as a mechanism involved with systemic cell migration. The resultsprovide a non-invasive method for the detection of HGF in nasal mucus.

TABLE 25 HGF in human plasma, urine, saliva and nasal mucus HGF PROTEINHGF BIOLOGICAL FLUIDS (17) μg/ml mg/dl PROTEIN PLASMA 709 ± 91^(†) 3.1 ±0.1 100 ± 13   URINE  623 ± 126 — — SALIVA 0 0 NASAL MUCUS 2015 ±431^(a) 2.2 ± 0.1 924 ± 227^(a) ( ) Subject number ^(†)MEAN ± SEM Withrespect to plasma and urine ^(a)p < 0.001

Example 26 Analysis of Platelet Derived Growth Factor (PDGF) in HumanPlasma, Urine, Saliva and Nasal Mucus

Table 26 illustrates detection and measurement of PDGF in human plasma,urine, saliva and nasal mucus in 18 subjects. Concentrations of PDGFexpressed per mg protein were found to be higher in saliva and nasalmucus than in plasma. These results suggest that PDGF may be synthesizedin the serous glands of the nose for a specific mechanism involved withnasal homeostasis. The results provide a non-invasive method for thedetection of PDGF in nasal mucus.

TABLE 26 PDGF in human plasma, urine, saliva and nasal mucus PDGFPROTEIN PDGF BIOLOGICAL FLUIDS (18) μg/dl mg/dl PROTEIN PLASMA  510 ±153^(†) 6.9 ± 0.1  71 ± 21 URINE 5 ± 2 — — SALIVA 600 ± 176 2.6 ± 0.2215 ± 18^(a) NASAL MUCUS 482 ± 87  3.0 ± 0.2 175 ± 32^(b) ( ) Subjectnumber ^(†)MEAN ± SEM ^(a)p < 0.001 with respect to plasma ^(b)p < 0.02with respect to plasma

Example 27 Analysis of Carbonic Anhydrase VI (CA VI) Concentration

Table 27 illustrates decrease in CA VI in patients with smell and tasteloss. These results provide a method for the detection and measurementof CA VI in nasal mucus as an index of smell and taste loss and itscontinual measurement during treatment of these disorders in order tomonitor efficacy of therapy.

TABLE 27 CA VI concentrations in nasal mucus in normal subjects and inpatients with smell loss SUB- PROTEIN ZINC COPPER CA VI JECTS mg/dl μg/Lμg/L mg/ml NOR- (8) 3.41 ± 0.02^(†) 97 ± 8 102 ± 8  0.287 ± 0.056 MALSMEN (5) 2.96 ± 0.30  100 ± 11  78 ± 26 0.238 ± 0.03  WOMEN (3) 3.54 ±2.04  90 ± 8 103 ± 15 0.369 ± 0.26  PA- (70) 2.27 ± 0.04  0.157 ± 0.13 TIENTS MEN (39) 2.21 ± 0.14^(a) 182 ± 17 118 ± 14 0.158 ± 0.020 WOMEN(31) 2.34 ± 0.18^(a) 171 ± 21 126 ± 12 0.155 ± 0.014 ( ) Subject number^(†)Mean ± SEM Compared to normals ^(a)p < 0.001

Example 28 Analysis of Loss of Smell Function by Disease Etiology

Table 28 illustrates loss of smell function by disease etiology withrespect to measurements of CA VI concentration in nasal mucus. Resultsindicate that patients with post influenza hyposmia hypogeusia (PIHH),allergic rhinitis and post anesthesia have significantly decreased CA VIconcentrations in nasal mucus. These results provide a method for thedetection and measurement of CA VI in nasal mucus as an index of smelland taste loss and its continual measurement during treatment of thesedisorders in order to monitor efficacy of therapy. The detection of CAVI in nasal mucus provides a non invasive method of diagnosing variousdiseases related to human physiology and pathology.

TABLE 28 Carbonic Anhydrase VI concentrations in nasal mucus in normalsubjects and in patients with smell loss PROTEIN ZINC COPPER CA VICONDITION mg/dl μg/L μg/L μg/ml NORMALS (11)  3.17 ± 0.48^(†) 97 ± 7 102± 7  0.287 ± 0.044 PIHH (26) 2.39 ± 0.19  139 ± 18^(a)  105 ± 11^(a)0.186 ± 0.02^(c ) ALLERGIC RHINITIS (25) 2.34 ± 0.19  234 ± 24^(a) 139 ±20  0.141 ± 0.018^(b) POST ANESTHESIA (6)  1.65 ± 0.30^(b) 189 ± 60 139± 40  0.156 ± 0.047^(c) PHANTAGEUSIA (5) 2.30 ± 0.59 170 ± 49 158 ± 310.180 ± 0.054 ( ) Subject number ^(†)Mean ± SEM Compared to normals^(a)p < 0.001 ^(b)p < 0.025 ^(c)p < 0.05

Example 29 Analysis of cAMP and cGMP in Human Nasal Mucus and in ParotidSaliva

Table 29 illustrates detection and measurement of cAMP and cGMP insaliva and in nasal mucus in normal subjects. Results show that patientswith smell loss had decreased levels of cAMP in their nasal mucus. Theseresults indicate that cAMP in nasal mucus can be an index of smell lossand that its secretion may be inhibited in smell loss. Thus, monitoringof cAMP in the nasal mucus can be a diagnostic tool for the treatment ofdiseases related to cAMP. Results also indicate that there was lesssignificant difference between cGMP in human nasal mucus and in parotidsaliva. The results provide a non-invasive method for the detection ofcAMP and cGMP in nasal mucus.

TABLE 29 Comparison of cAMP and cGMP in human nasal mucus and in parotidsaliva NASAL MUCUS PAROTID SALIVA^(††) TOTAL cAMP*   0.22 ± 0.07^(†$)2.00 ± 0.31 cGMP 0.25 ± 0.08 0.21 ± 0.04 MEN cAMP  0.21 ± 0.13^($) 1.58± 0.43 cGMP 0.28 ± 0.16 0.23 ± 0.06 WOMEN cAMP  0.23 ± 0.06^($) 3.38 ±0.35 cGMP 0.24 ± 0.13 0.20 ± 0.07 PROTEIN** TOTAL 3.24 ± 0.22 3.11 ±0.18 MEN 3.32 ± 0.02 3.39 ± 0.34 WOMAN 3.51 ± 0.75 2.93 ± 0.02 *inpmol/ml **mg/ml ^(†)MEAN ± SEM ^(††)Collected in 171 subjects ^($)p <0.001 compared to parotid saliva

Example 30 Analysis of cAMP and cGMP in Human Nasal Mucus in NormalSubjects and in Patients

Table 30 illustrates comparison of the measurement of cAMP and cGMP innormal subjects with the patients with taste and smell loss. Resultsshow that patients with smell loss had decreased levels of cAMP in theirnasal mucus. These results indicate that cAMP in nasal mucus can be anindex of smell loss and that its secretion may be inhibited in smellloss. Thus, monitoring of cAMP in the nasal mucus can be a diagnostictool for the treatment of diseases related to cAMP. Results alsoindicate that there was less significant difference between cGMP innasal mucus in normal subjects or in patients with hyposmia.

TABLE 30 Comparison of cAMP and cGMP in human nasal mucus in normalsubjects and in patients with smell loss (Hyposmia) cAMP cGMP pmol/mgpmol/mg PROTEIN pmol/ml protein pmol/ml protein mg/min CONDITION  (41)0.22 ± 0.02^(†) 0.08 ± 0.01 0.25 ± 0.04 0.06 ± 0.01 3.37 ± 0.19  NORMALMEN  (34) 0.21 ± 0.01   0.05 ± 0.01 0.28 ± 0.02  0.04 ± 0.004 3.32 ±0.13  WOMEN  (7) 0.23 ± 0.06   0.10 ± 0.04 0.24 ± 0.13 0.10 ± 0.04 3.59± 1.02  PATIENTS (146) 0.14 ± 0.02^(d) 0.07 ± 0.01 0.20 ± 0.02 0.12 ±0.01 2.48 ± 0.08^(b) MEN  (63) 0.14 ± 0.02^(c) 0.07 ± 0.02 0.25 ± 0.030.12 ± 0.02 2.50 ± 0.13^(b) WOMEN  (83) 0.15 ± 0.02   0.07 ± 0.01 0.17 ±0.02 0.11 ± 0.01 2.46 ± 0.11^(c) ( )Subject number ^(†)Mean ± SEM ^(b)p< 0.005 compared to normals ^(c)p < 0.01 compared to normals ^(d)p <0.05 compared to normals

Example 31 Analysis of cAMP and cGMP Concentrations in Nasal Mucus ofPatients

Table 31 illustrates detection and measurement of cAMP and cGMPsecretion in nasal mucus in patients with graded severity of smell loss(anosmia<Type I hyposmia<Type II hyposmia from most severe to leastsevere). Data indicates that as degree of smell loss increased, levelsof cAMP in nasal mucus decreased. These data confirm the relationshipbetween cAMP secretion in nasal mucus and degree of smell loss. Resultsalso indicate that there was less significant difference between cGMP innasal mucus in normal subjects or in patients with hyposmia.

TABLE 31 cAMP and cGMP concentrations in nasal mucus in patients withvarious degrees of smell loss TOTAL cAMP cGMP PROTEIN cAMP PROTEIN cGMPPROTEIN PATIENTS mg/ml pmol/ml pmol/mg pmol/ml pmol/mg ANOSMIA  (2) 1.410.004 0.003 0.179 0.127 HYPOSMIA TYPE I (17) 2.61 ± 0.29  0.116 ± 0.04*0.034 ± 0.01  0.225 ± 0.05 0.101 ± 0.02 TYPE II (64) 2.56 ± 0.13 0.193 ±0.03 0.102 ± 0.02^(a) 0.189 ± 0.03 0.079 ± 0.01 TYPE III NORMAL (10)3.70 ± 0.67 0.225 ± 0.67 0.088 ± 0.04  0.356 ± 0.13 0.094 ± 0.03SUBJECTS ( )Patient number *Mean ± SEM ^(a)p < 0.001 with respect toType I hyposmia

Example 32 Analysis of Nitric Oxide (NO) in Saliva and in Nasal Mucus

Table 32 illustrates detection and measurement of NO in human saliva andnasal mucus. NO was found to be present in both saliva and nasal mucusand its mean concentration in saliva were 21% lower in patients than innormal subjects whereas in nasal mucus mean levels were 25% lower inpatients. Treatment which increases cAMP in nasal mucus and improvessmell function may be mirrored by increases in nasal mucus NO. Thedetection of NO in nasal mucus provides a non invasive method ofdiagnosing various diseases related to human physiology and pathology.

TABLE 32 NO in saliva and nasal mucus in normal subjects and in patientswith smell loss SALIVA NASAL MUCUS NO PROTEIN NO/ NO PROTEIN NO/SUBJECTS μg/ml mg/dl PROTEIN μg/ml mg/dl PROTEIN NORMALS (15) 0.57 ±0.03^(†) 2.6 ± 0.1 0.23 ± 0.02 0.48 ± 0.08 2.3 ± 0.15 0.22 ± 0.05PATIENTS (34) 0.45 ± 0.06   2.8 ± 0.1 0.18 ± 0.03 0.36 ± 0.03 2.0 ± 0.080.21 ± 0.03 ( )Subject number ^(†)MEAN ± SEM

Example 33 Analysis of Nitric Oxide (NO) in Nasal Mucus in PatientsBefore and after Theophylline Treatment

Table 33 illustrates NO levels in nasal mucus in patients treated withTheophylline in various doses before and after drug treatment. NO levelsin nasal mucus changed following the treatment of patients with smellloss. Results show treatment of patients with graded increasing doses oftheophylline and measurement of both smell function and NO in nasalmucus in patients with hyposmia. Results indicated that prior to thetreatment levels of NO in nasal mucus were lower than in normalsubjects. After treatment with theophylline in graded doses there wereincreases in nasal mucus NO associated with graded increases in smellfunction. These data demonstrate that treatment with drugs that increasesmell function to or toward normal, returns smell function to normal.These results also demonstrate the measurements of various substances innasal mucus as an index of both human physiology and pathology ofvarious diseases. Its continual measurement during treatment of thesedisorders helps in monitoring efficacy of therapy. The detection of NOin nasal mucus provides a non invasive method of diagnosing variousdiseases related to human physiology and pathology.

TABLE 33 NO in nasal mucus in patients with Theophylline in variousdoses before and after drug treatment PRE- TREATMENT 200 mg 400 mg 600mg NO Protein NO Protein NO Protein NO Protein SUBJECTS μg/ml mg/dlμg/ml mg/dl μg/ml mg/dl μg/ml mg/dl Patients (12) 0.35 ± 0.07 1.6 ± 0.30.25 ± 0.01 1.7 ± 0.3 0.40 ± 0.06 2.1 ± 0.10 0.59 ± 0.16 1.9 ± 0.15Normal 0.61 ± 0.20 ( )Subject number

Example 34 Analysis of Insulin Like Growth Factor (IGF 1) in HumanSaliva and Nasal Mucus

Table 34 illustrates detection and measurement of IGF 1 in human salivaand nasal mucus in 26 subjects. Results show that IGF 1 concentration innasal mucus was significantly greater than in saliva. Results indicatethat the measurement of nasal mucus IGF 1 can be used as an index ofhuman physiology and pathology. The detection of IGF 1 in nasal mucusprovides a non invasive method of diagnosing various diseases related tohuman physiology and pathology.

TABLE 34 IGF 1 in human saliva and nasal mucus SALIVA NASAL MUCUS IGF 1PROTEIN IGF 1/ IGF 1 PROTEIN IGF 1/ μg/ml mg/dl PROTEIN μg/ml mg/dlPROTEIN SUBJECTS (26) 11.4 ± 0.5^(†) 3.1 ± 0.2 4.6 ± 0.4 13.7 ± 0.4^(b)2.2 ± 0.2 8.9 ± 1.0^(a) ( )Subject number ^(†)MEAN ± SEM With respect tosaliva ^(b)p < 0.005 ^(a)p < 0.001

Example 35 Analysis of TNF∂, TNFR₁, and TNFR₂, in Nasal Mucus ofPatients

Table 35 illustrates detection and measurement of TNFα, and soluble TNFreceptors 1 and 2, moieties in the nasal mucus. TNFα, and soluble TNFreceptors 1 and 2 increase in systems undergoing excessive apoptosis.Treatment with 600 mg theophylline restored smell function to or towardnormal in these patients. There was a significant dose-response decreasein each moiety related to a stepwise increase in theophylline treatmentassociated and a stepwise improvement in olfactory acuity. These resultssuggest that pathological apoptosis of olfactory epithelial anatomycauses smell loss in patients with hyposmia; this process is reversedwith theophylline therapy which restores smell function in thesepatients. These results illustrate biochemical parameters associatedwith return of smell function in patients treated with theophylline.

TABLE 35 TNF∝, TNFR₁, TNFR₂, in nasal mucus in patients with hyposmiatreated with Theophylline Theophylline treatment (mg/l) PATIENTS PRE 200400 600 TNF∝* (24)  18.3 ± 6.1^(†)  20.0 ± 2.8  12.1 ± 2.1^(d)  7.4 ±1.8^(a) TNFR₁ (19) 2,353 ± 718 3,148 ± 663 1,146 ± 220^(b) 1,220 ±286^(ab) TNFR₂ (18) 1,747 ± 535 1,952 ± 339   949 ± 180^(c)   916 ± 344*in pg/ml ( ) Subject Number ^(†)Mean ± SEM ^(a)p < 0.001 with respectto 200 mg. ^(b)p < 0.001 with respect to 200 mg. ^(c)p < 0.025 withrespect to 200 mg. ^(d)p < 0.05 with respect to 200 mg.

Example 36 Analysis of TNF∝ in Nasal Mucus in Patients Before and afterTreatment with Theophylline

These studies were extended to reveal levels of TNFα in nasal mucus ofpatients with graded loss of smell following treatment with theophylline(Table 36). Smell loss was graded such that loss was greatest in Type Ihyposmia, less in Type II and still less in Type III (I>II>III).Pretreatment levels of TNFα were significantly higher in patients withType I hyposmia than in Type II hyposmia consistent with their greaterdegree of smell loss. Following treatment which was effective inrestoring smell function toward normal, levels of TNFα decreased in eachpatient group consistent with each increased dose of theophylline whichgenerated a dose dependent increase in smell function—as theophyllinedose increased, smell function increased and TNFα levels decreased. InType I hyposmia TNFα decreased 40% after treatment with 400 mgtheophylline and 57% after treatment 600 mg. In Type II hyposmia TNFαdecreased 11% after treatment with 600 mg theophylline.

TABLE 36 TNF∝ in nasal mucus in patients with various types of hyposmiatreated with theophylline Theophylline treatment* HYPOSMIA TYPE PRE 200400 600 I (8) 26.0 ± 6.5^(tb) — 15.6 11.2 ± 4.4 II (13)  5.3 ± 1.0 14.9± 2.5 11.5 ± 3.2  4.3 ± 0.8^(a) III (3) —  1.2 ± 0.7^(bc) *in mg orallydaily ( ) Subject Number ^(t)Mean ± SEM ^(a)p < 0.001 with respect to200 mg ^(b)p < 0.001 with respect to Type I (600 mg) ^(c)p < 0.01 withrespect to Type II (600 mg) ^(d)p < 0.001 with respect to Type II, pretreatment

Example 37 Analysis of TNFR 1 in Nasal Mucus in Patients Before andafter Treatment with Theophylline

TNF Receptor 1 (TNFR1) exhibited similar results in nasal mucus inpatients with smell loss after treatment with theophylline (Table 37).With increased smell loss pre treatment TNFR 1 levels were increased innasal mucus in patients with Type I hyposmia (whose smell loss wasincreased over that of patients with Type II hyposmia). Aftertheophylline treatment, as a dose of drug increased, TNFR 1 levelsdecreased in Type I associated with increases in smell function. Levelsof TNFR 1 at 600 mg theophylline were systematically decreased inrelation to degree of smell loss. The greater was the smell loss, thehigher was the level of TNFR 1.

TABLE 37 TNFR 1 in nasal mucus in patients with various types ofhyposmia treated with theophylline Theophylline treatment* HYPOSMIA TYPEPRE 200 400 600 I (7) 4,626 ± 1,647^(tb) 6,521 ± 1,304^(c) 1,498 1,832 ±704^(a)  II (10)   837 ± 60 1,462 ± 371 1,087 ± 335 862 ± 335 III (2) —— — 585 ± 335 *in mg orally daily ( ) Subject Number ^(t)Mean ± SEM^(a)p < 0.005 with respect to 200 mg ^(b)p < 0.02 with respect to TypeII pre treatment ^(c)p < 0.01 with respect to Type II or 200 mg

Example 38 Analysis of TNFR 2 in Nasal Mucus in Patients Before andafter Treatment with Theophylline

TNR Receptor 2 (TNFR 2) in nasal mucus exhibited similar results (Table38). Pretreatment with theophylline in patients with the most severesmell loss (Type I hyposmia) exhibited higher TNFR 2 levels in nasalmucus than did patients with less severe smell loss (Type I hyposmia).With a dose dependent increase of theophylline treatment levels of TNFR2 in nasal mucus decreased consistent with a dose dependent increase insmell function. Levels of TNFR 2 in patients with Type I and Type IIhyposmia decreased almost 50% on 600 mg theophylline consistent withtheir greatest return of smell function. At this highest level oftheophylline levels of TNFR 2 were decreased in relationship to thedecrease in smell function—TNFR 2, Type I>Type II>Type III; smell loss,Type I>Type II>Type III.

TABLE 38 TNFR 2 in nasal mucus in patients with various types ofhyposmia treated with theophylline Theophylline treatment* HYPOSMIA TYPEPRE 200 400 600 I (7) 2,718 ± 1,125^(t) 3,100 ± 1,184 1,491 ± 1,102 II(10) 1,145 ± 625    1,378 ± 480   1,014 ± 272 553 ± 158 III (2) — — —436 ± 443 *in mg orally daily ( ) Subject Number ^(t)Mean ± SEM ^(a)p <0.001 with respect to 200 mg ^(b)p < 0.001 with respect to Type I (600mg) ^(c)p < 0.01 with respect to Type II (600 mg)

Example 39 Analysis of Endoglin in Human Plasma, Urine, Saliva and NasalMucus

Table 39 illustrates detection and measurement of Endoglin in the nasalmucus. Results indicate that the measurement of nasal mucus Endoglin canbe used as an index of human physiology and pathology. The detection ofEndoglin in nasal mucus provides a non invasive method of diagnosingvarious diseases related to human physiology and pathology.

TABLE 39 Endoglin in human plasma, urine, saliva and nasal mucus PLASMAURINE SALIVA NASAL MUCUS ENDOGLIN PROTEIN ENDOGLIN ENDOGLIN ENDOGLINENDOGLIN PROTEIN ENDOGLIN SUBJECTS mg/ml mg/dl PROTEIN mg/ml mg/ml mg/mlmg/dl PROTEIN (33) 2.7 ± 0.1^(†a) 7.1 ± 0.1 0.38 ± 0.1 0 0 0.2 ± 0.1 2.8± 0.1 0.07 ± 0.01 ( )Subject number ^(†)MEAN ± SEM ^(a)p < 0.001 withrespect to nasal mucus

Example 40 Increased Carbonic Anhydrase (CA) I, II and VI, Zinc andCopper after rTCMS

Ninety three patients, aged 18-85 y (52±2 y, Mean±SEM), 49 men, aged29-74 y (51±3 y) and 44 women, aged 20-85 y (53±3 y) with hyposmia,hypogeusia, and subsequent phantageusia (distortion of taste independentof any oral stimulus) and/or phantosmia (distortion of smell independentof any environmental odor) were studied before and after rTCMS in asingle blind placebo controlled fixed sequence clinical trial.

Patient symptoms persisted for 0.4-30 y (6.9±1.5 y) prior to rTCMS.Physical examination of head and neck including examination of oral andnasal cavities (the nose examined by anterior rhinoscopy with use ofvasoconstrictor agents) was within normal limits. Neither neurologicalnor psychiatric abnormalities other than taste and/or smell dysfunctionwas present in any patient. Anatomical magnetic resonance imaging (MRI)of brain and electroencephalographic (EEG) studies in all patients werewithin normal limits.

rTCMS was performed with a Cadwell magnetic pulse stimulator (Kennewick,Wash.) monitored with a TECA TD20 (Pleasantville, N.Y.) wave formgenerator, as described in Cicinelli, P., et al., Eletroenceph. Clin.Neurophys, 1997, 105:438-450; Henkin, Robert, et al., FASEB J., 2002,16:A878; Henkin, R. I., Encyclopedia of Neuroscience (3^(rd) Ed),Adelman, G. Smith, B H eds, Birkhauser, Boston, 2003, and; Moharram, R.,et al., FASEB J., 2004, 18:A201, all incorporated by reference in theirentirety herein.

Stimulation was applied in a fixed manner to four skull locations (leftand right temporoparietal, occipital, frontal of patients). Stimulusfrequency was 1 pulse given per 1-3 sec for 30-90 sec with 20 pulsesgiven at each location. Repeat stimulation was performed in all patientsin whom increased sensory acuity and/or decreased sensory distortionsoccurred; repetition continued (two-six applications) until no furtherincrease in sensory acuity and/or decrease in sensory distortionsoccurred.

One hour before and one to two hr after completion of rTCMS, venousblood and parotid saliva were collected. Whole venous blood was placedinto zinc free tubes containing 100 μl of zinc free heparin, on ice,centrifuged at 3000 rpm at 4° C., plasma removed and stored at −20° C.until assayed. Erythrocytes were washed and treated as described inAgarwal, R. P., et al., Bio. Tr. Elem. Res., 1985, 7:199-208,incorporated by reference in its entirety herein. Parotid saliva wascollected in four plastic tubes on ice using a modified Lashley cupapplied to Stensen's duct with maximal stimulation using reconstitutedlemon juice applied to the lingual surface as described in, Henkin, R.I., et al., Proc. Nat. Acad. Sci. USA, 1975, 72:488-492 and Henkin, R.I., et al., Amer. J. Med. Sci., 1976, 272:285-299, both incorporated byreference in their entirety herein. The first three tubes were collectedat two min intervals, the fourth tube until approximately eight ml wascollected. For convenience only results of saliva from the fourth tubewill be presented. Five hundred μl of saliva from the fourth tube wasplaced in dry ice immediately after collection and stored at −60° C.until measurements by SELDI-TOF mass analysis was performed.

Zinc and copper were measured in each tissue by atomic absorptionspectrophotometry (AAS) by using a double beam ThermoJarrell Ash video22 (Franklin, Mass.) AAS modified by the Maxwell Instrument Company(Salisbury, N.C.), the methods previously described in, Henkin, R. I.,et al., Amer. J. Med. Sci, 1999, 318, 380-391; Agarwal, R. P., et al.,Bio. Tr. Elem. Res., 1985, 7:199-208; Henkin, R. I., et al., Amer. J.Med. Sci., 1976, 272:285-299, and; Meret, S., et al., Clin. Chem., 1971,17:369-373, all incorporated by reference in their entirety herein.Saliva protein was determined by measurement of total peptide content byuse of absorbance at A215-A225 (called Δ215) and the extinctioncoefficient. CA activity was measured by a modification of the method ofRichli, E. E., et al., J. Biol. Chem., 1964, 239:1065-1078, incorporatedherein by reference in its entirety. Saliva samples stored at −60° C.were thawed and 1 μl directly spotted on an H4 Protein Chip array (prewashed with 0.1% TFA in 50% aqueous acetonitrile) and their proteinprofile examined on a Ciphergen (Fremont, Calif.) PBS IIc mass analyzer.

Samples were first incubated in a humid chamber for 5-10 minutes at roomtemperature, then washed with 5% aqueous acetonitrile, dried and 1matrix added (sinapinic acid in 0.1% TFA, 50% aqueous acetonitrile).Samples were allowed to dry again and subjected to SELDI-TOF analysis onthe PBS IIc. Protein peaks were characterized by their apparentmolecular weight based on their mass/charge ratio (m/z).

Following initial observation of biochemical changes after rTCMSsubsequent measurements in all biological fluids were performed in ablinded manner; all samples were coded and results uncoded only afterall analyses were completed. Mean±SEM for each parameter was determinedfor each condition pre and post rTCMS. Differences between eachcondition were calculated for each parameter and significance ofdifferences determined by parametric (differences betweenundifferentiated means, paired t tests, X²) and non parametric (signtest) statistics.

Results:

After rTCMS mean CA VI activity and salivary zinc and copperconcentrations increased significantly as did mean CA I, II activity andplasma copper concentrations (Table 40). Significant increases in bothCA I, II and CA VI activity were also measured using paired comparisons(p<0.01 Student t test) and the sign test (p<0.05, Student t test) (datanot shown). These latter data are reflected in results shown in Table 41in which changes pre and post rTCMS are shown. Increased CA VI wasmeasured in 87% of patients with changes varying from −5% to +153% (meanchange, +17%) (Table 41); compared to chance changes of this magnitudewould occur <5 times in 1000 (X²). Increased CA I, II was measured in93% of patients with changes varying from −2% to +56% (mean change,+11%) (Table 41); compared to chance changes of this magnitude wouldalso occur <1 time in 1000 (X²). Increased plasma and erythrocyte zincand copper concentrations were measured in 91-93% of patients (Table41); compared to chance changes of this magnitude would also occur <1time in 1000 (X²).

TABLE 40 Changes in plasma, erythrocyte and saliva CA I, II and VI, zincand copper before (pre) and after (post) rTCMS in 93 patients rTCMS PREPOST CONDITION PLASMA Zn (μg/dl) 85 ± 2*   88 ± 2 Cu (μg/dl) 101 ± 2   109 ± 3^(a) ERYTHROCYTES Zn (μg/gHb) 38.0 ± 0.5   39.7 ± 0.5^(b) Cu(μg/gHb)  2.1 ± 0.04  2.2 ± 0.05 CA I, II (μg/g protein) 3.12 ± 0.06 3.45 ± 0.06^(d) SALIVA Zn (μg/L) 103 ± 5    121 ± 5^(b) Cu (μg/L) 13 ±1    22 ± 3^(c) CA VI (μg/g protein) 0.153 ± 0.009 0.197 ± 0.008^(d)*Mean ± SEM CA, carbonic anhydrase Compared to pre rTCMS ^(a)p < 0.05^(b)p < 0.025 ^(c)p < 0.01 ^(d)p < 0.001

TABLE 41 Changes in plasma, erythrocyte and saliva carbonic anhydrase I,II and VI, zinc and copper before (pre) and after repetitivetranscranial magnetic stimulations (rtcms) in patients with decreasedsensory acuity and presence of sensory distortions rTCMS PRE vs. POSTNUMBER CONDITION INCREASED DECREASED UNCHANGED INCREASED PLASMA % Zn(μg/dl) 61 6 0 91 Cu (μg/dl) 62 5 0 93 ERYTHROCYTES Zn (μg/gHb) 66 1 099 Cu (μg/gHb) 62 4 1 93 CA I, II (μg/g 62 4 1 93 protein) SALIVA Zn(μg/L) 49 18 0 73 Cu ((μg/L) 51 16 0 76 CA VI (μg/g 60 5 2 90 protein)

SELDI-TOF mass spectrometry revealed a peak at m/z 21.5K in the postrTCMS spectra which was absent in the pre rTCMS spectra (FIG. 10). Alsopresent in the post rTCMS spectra was a repetitive protein patternseparated by intervals of approximately 5K m/z (FIG. 10). Similarpatterns were observed in about ⅓ of patients studied.

Thus, the present study illustrates that several salivary proteins canbe induced after rTCMS (FIG. 10). Increased enzyme activities and zincand copper concentrations usually persisted two-four wk after rTCMS;however, over time there was a slow, gradual decrease in enzymeactivities and in plasma, erythrocyte and saliva zinc and copperconcentrations as well as a loss of sensory acuity and subsequent returnof sensory distortions.

This study demonstrates that biochemical changes may occur after rTCMS.Since changes in taste and smell function occur in several neurologicaldisorders these results may also relate to other conditions such asepilepsy, Parkinsonism, Alzheimer disease, head injury, and motor neurondisease in which rTCMS can be an effective therapeutic agent.

Example 41 Recovery of Taste and Smell Function Following rTCMS

Seventeen right handed Caucasian patients, five men, aged 40-′74 y (58±7y, X±SEM), 12 women, aged 30-′76 y (51±5 y) were studied. Each had mildto severe persistent hyposmia and hypogeusia as well as mild to severepersistent birhinal phantosmia and/or global oral phantageusia; thesensory distortions were profound enough to interfere with normal lifepursuits. Acuity loss persisted for 6 mo to 30 y (4.1±2 y) and sensorydistortions persisted for 3 mo to 30 y (3.7±2 y) prior to clinic visit.Etiologies which initiated their symptoms were head injury (4 patients),post influenza-like infection (PIHH, 7 patients), idiopathic causes (4patients) and drug reactions (2 patients). Each of the 17 patients whopresented with these symptoms was treated with rTCMS.

None had any neurological symptom other than loss of sensory acuity andpresence of sensory distortions. None had any psychiatric symptom otherthan some depression associated with persistence of these cognitiveimpairments. Symptoms were unrelieved by any prior treatment withmultiple agents including antiepileptics, anxiolytics, antidepressants,trace metals, vitamins and a variety of alternative treatment modalitiesincluding herbal remedies, acupuncture, chiropractic techniques andhypnosis. Physical examination of each patient, including examination ofthe head and neck and general neurological examination, was withinnormal limits. Both anatomical brain MRI and electroencephalograms werewithin normal limits in each patient.

Measurement techniques: A battery of tests measuring taste and smellacuity and character and degree of sensory distortions were administeredto each patient. Taste and smell acuity were determined by standardthree stimuli forced choice staircase techniques (Henkin R. I., Amer. J.Med. Sci. 1976, 272:285-299, incorporated herein by reference in itsentirety) by which detection (DT) and recognition (RT) thresholds andmagnitude estimation (ME) for four tastants [NaCl (salt), sucrose(sweet), HCl (sour) and urea (bitter)] and four odorants [pyridine (deadfish), nitrobenzene (bitter almond), thiophene (old motor oil) and amylacetate (banana oil)] were determined, and reference values establishedfor a large number of normal subjects. Results for DT and RT, in mmol/Land M/L for tastants and odorants, respectively, were converted intobottle units (BU) and compared to previously established standards.Magnitude estimation (ME) was determined, calculated in % for eachstimulus and compared to previously described standards in Henkin et al.Otolaryngology, 1993, vol. 2, p 1-86 and Henkin et al. Drug safety,11:310-377, 1994, incorporated herein by reference in its entirety.

Sensory distortions were graded daily in intensity, duration andfrequency using a written record on a 0-100 scale for 3 d-4 wk prior torTCMS; 0 reflected total absence of sensory distortions, 100 reflectedthe digitized composite of the most intense distortion experienced overthe entire day. Records were reviewed prior to the study to insureadequate understanding of symptom grading. The entire battery ofcognitive measurements was obtained at the initial patient visit andrepeated immediately prior to and after each rTCMS treatment. Thisbattery was also repeated at variable intervals (1 day, 2-4 wk, 6-46 mo)after each rTCMS treatment. Each test battery was performed independentof knowledge of any prior test result.

Treatment protocol: rTCMS was performed with a Cadwell magnetic pulsestimulator (Kennewick, Wash.) monitored by a TECA TD20 (Pleasantville,N.Y.) wave form generator. Stimulation was applied by a single openquadrangular 12×12 cm coil. Three sequential stimulation protocols wereused. The first two were considered placebo or sham trials.

For the initial sham trial, 20 stimuli at intervals of 1-3 sec wereapplied sequentially at the lateral acromial process of the clavicle(near Erb's point) (a) to the anterior right shoulder, then (b) anteriorleft shoulder at 20-30% maximal output [20-30% of 1.5 T or ˜0.2-0.4 T(since stimulus delivery was non-linear)] and then (c) to the back ofthe mid neck region (at the level of C5-8 at 30-40% maximal output or˜0.4-0.8 T); mild muscle group flexion of arm and hand muscles (shoulderstimulation) and neck, strap and facial muscles (neck) followed eachstimulation and was visually monitored.

For the second sham trial, 20 stimuli at intervals of 1-3 sec wereapplied sequentially to four skull regions (left temporoparietal,occipital, right temporoparietal, frontal) at 20% maximal output; thiswas considered subthreshold stimulation since no peripheral muscleresponses occurred.

For the treatment trial, 20 stimuli at intervals of 1-3 sec were appliedat 40-55% maximal output (˜0.8-LIT) to each skull location as in thesecond sham trial noted above. Muscle responses to this latterstimulation were present and monitored by visual observation (e.g.,right/left thenar and/or phalangeal flexion with left/righttemporoparietal stimulation, respectively).

After each 20 stimuli of sham or treatment stimulation at eachanatomical location, olfactory response to presentation of a single odor(one concentration of an odorant whose DT, RT and ME were previouslydetermined) and/or changes in intensity and character of phantageusiaand/or phantosmia (previously determined) was recorded. If any change inolfactory acuity or in sensory distortion occurred after anystimulation, stimulation at that location at that same intensity wasrepeated two-six times until no further change occurred.

Outcome measures: Mean±SEM of changes in taste and smell acuity (DT, RT,ME) and in intensity of sensory distortions before and after each rTCMStreatment were calculated and significance of differences determined byStudent's t tests. Differences were also calculated using paired t testswith significance of differences pre and post rTCMS determined byStudent's t test.

Results: Pre rTCMS I

Taste: Mean DTs and RTs for all tastants were above normal (i.e. acuitywas decreased). Mean MEs for all tastants were below normal (i.e. acuitywas decreased) (Table 42). Mean DT and RT for all tastants except DT forHCl were significantly above normal and mean MEs for all tastants weresignificantly below normal.

Smell: Mean DTs and RTs for all odorants were above normal (i.e. acuitywas decreased) and mean MEs for all odorants were below normal (i.e.acuity was decreased) (Table 42). Mean DT and RT for all odorants(except DTs for pyridine, thiophene and amyl acetate) and mean MEs ofall odorants were significantly above normal (Table 42).

Sensory distortions: Phantageusia intensity was 82±7%. Phantosmiaintensity was 72±14% (Table 43). There were no gender differences ineither phantageusia or phantosmia intensity (Table 43).

Results: Post rTCMS I

Placebo or sham stimulation (0.2-0.4 T): No subjective or objectivechanges in either taste and/or smell acuity or in character or intensityof sensory distortions occurred in any patient following stimulation ofshoulders or neck or in any skull location.

Treatment Stimulation (0.8-1.1 T):

Taste: Mean DTs and RTs for all tastants decreased (i.e., acuityincreased) and mean MEs for all tastants increased (i.e. acuityincreased) (Table 42). Mean DT and RT returned to normal levels forNaCl, sucrose and HCl as did DT for urea and ME for all tastants (Table42). Only mean RT for urea did not return to normal although it wassignificantly lower than before treatment (Table 42).

Smell: Mean DTs and RTs for all odorants decreased (i.e., acuityincreased) and mean MEs for all odorants increased (i.e., acuityincreased) (Table 42). Mean DT and RT for pyridine, nitrobenzene andthiophene and mean DT for amyl acetate returned to or below normallevels after treatment (Table 42). Only mean RT for amyl acetate did notreturn to normal although it was significantly below values obtainedbefore this treatment. Mean ME for all odorants also returned to normallevels.

Sensory distortions: Mean phantageusia and phantosmia intensitydecreased significantly (Table 43). In each man phantosmia disappeared.

Response Summary: No changes in taste or smell acuity or in sensorydistortion intensity occurred in two patients immediately aftertreatment stimulation [(one with head injury, one with PIHH, both women,data included in Tables 42 and 43)]. These two patients were labelednon-responders. Reports of no change in sensory distortion intensity andno change in repeat acuity testing occurred in these two patients 2-7 dafter treatment. No changes were reported 4 wk after rTCMS I and nofurther data about these patients were obtained.

Taste and smell acuity returned to normal levels for all tastants andodorants and all sensory distortions completely disappeared in twopatients within one hr after rTCMS I [(one with head injury (one woman)one with PIHH (one man), data included in Tables 42 and 43)]. These twopatients were labeled responders. Repeat testing 2-7 d after rTCMS Idemonstrated normal sensory acuity and no sensory distortions werereported (data not shown). Reports of normal sensory acuity and absenceof any sensory distortions were received for as long as these patientswere followed (46 mo).

In the remaining 13 patients sensory acuity improved (Table 42) andsensory distortions diminished (Table 43) one hour after stimulation(Table 43). These 13 patients were also labeled responders. Symptomimprovement occurred (vs) in all responders when the field was appliedat only one skull location. Seven patients reported improvement afterleft temporoparietal stimulation, four (27%) after right temporoparietalstimulation, three (20%) after frontal stimulation and one (7%) afteroccipital stimulation. There was no improvement in the non-responders nomatter where the field was applied.

Later Post rTCMS I

Four wk-2 mo after rTCMS I, repeat testing of taste and smell acuity andmeasurement of sensory distortion intensity indicated that cognitiveimpairments had returned in 13 of the 15 responders (vs). A second trialof rTCMS (rTCMS II) was instituted in these patients.

Pre rTCMS II

Immediately prior to rTCMS II, the entire battery of sensory tests andmeasurement of sensory distortion intensity previously measured wererepeated (Table 44).

Taste: Compared to immediately post rTCMS I, mean DTs and RTs for alltastants (except DT for HCl which did not change) increased (i.e.,acuity decreased) and mean MEs for all tastants decreased (i.e., acuitydecreased) (cf Tables 44, 42).

Smell: Compared to immediately post rTCMS I, mean DTs and RTs for allodorants (except RT for thiophene which was lower than post rTCMS I)increased (i.e., acuity decreased) and mean MEs for all odorantsdecreased (i.e., acuity decreased), (except for thiophene which washigher (cf Tables 44, 42). However, mean MEs were all higher than prerTCMS I indicating that some improvement after rTCMS I was retained.

Sensory distortions: Compared to immediately after rTCMS I, meanestimates of phantageusia intensity increased but were significantlyless than intensities measured prior to rTCMS I (cf Tables 45, 43, prerTCMS I, 82±7, later post rTCMS I, 39±20, p<0.05 t test, p<0.02 paired ttest). Similarly mean estimates of phantageusia intensity also increasedbut were less than pre rTCMS I (cf Tables 45, 43, pre rTCMS I, 72±14,later post rTCMS I, 47±8, p>0.05 t test, p<0.05 paired t test). Similarchanges also occurred in phantosmia (cf Tables 45, 43).

Response summary: These results suggest that the improvement incognitive impairments which occurred immediately after rTCMS I persistedto some extent but these patients “escaped” from this improvement with areturn of cognitive impairments. A second course of rTCMS wasinstituted.

rTCMS II

Placebo or sham stimulation (0.2-0.4 T): No subjective or objectivechanges in either taste and/or smell acuity or in character or intensityof sensory distortions occurred in any patient following stimulation ofshoulders, neck or in any skull location.

Treatment Stimulation (0.8-1.1 T):

Taste: Mean DTs and RTs for all tastants decreased (i.e., acuityincreased) and mean MEs for all tastants increased (i.e., acuityincreased) (Table 44) as after rTCMS I (Table 42). There weresignificant decreases in DTs for NaCl and urea and RTs for HCl and urea.Mean MEs for all tastants were not significantly different from normal(Table 44). DT for NaCl was significantly lower, (i.e., relativeincreased acuity) than normal as was DT for urea.

Smell: Mean DTs and RTs for all odorants decreased (i.e., acuityincreased) and mean MEs for all odorants increased (i.e., acuityincreased) (Table 44) as after rTCMS I (Table 42). There were nosignificant differences in mean DTs, RTs or MEs for any odorant withrespect to normal. DTs for nitrobenzene and thiophene and RTs forpyridine and nitrobenzene were lower than normal (i.e., relativeincreased acuity) and MEs for pyridine, nitrobenzene and thiophene werehigher than normal (i.e., relative increased acuity).

Sensory distortions: Significant decreases occurred in phantageusia andphantosmia (Table 45) as after rTCMS I (Table 43). Phantosmia completelydisappeared after treatment in all patients including all men and women,not just in men as after rTCMS I (Table 43). Phantageusia disappeared in10 patients, improved by 50% in one and only slightly, if at all, intwo. After rTCMS II both mean phantageusia and phantosmia intensitydecreased to levels below those measured later post rTCMS I (cf, Tables43, 45).

In these 13 patients rTCMS II was effective in initiating improvementagain only at the same locus at which initial improvement occurred afterrTCMS I.

Response Summary: After rTCMS II improvement lasted longer than afterrTCMS I, lasting wk-mo. In seven of these 13 (54%) (one with headinjury, four with PIHH, two idiopathic) return of sensory acuity tonormal and total cessation of sensory distortions persisted for as longas measurements were made (30 mo). In the remaining six patients (onewith head injury, one with PIHH, the two with idiopathic causes, the twowith drug reactions) symptoms of cognitive impairment returned afterone-five mo but acuity was less impaired and sensory distortions wereless intense than prior to rTCMS II (as in pre rTCMS I and II).

These six patients who “escaped” from rTCMS II were again treated withrTCMS (rTCMS III) as in rTCMS I and II. This therapy was again effectivein improving cognitive impairments but again only at the same locus thatinitiated improvement after prior stimulation (data not shown). Allsensory acuity returned to normal levels and all sensory distortionsdisappeared in these six patients for as long as they were followed(6-36 mos).

The results indicate that rTCMS was efficacious in improving tasteand/or smell acuity and in inhibiting phantosmia and phantageusia inmost patients who exhibited these symptoms. Improvement occurred afterrTCMS at one brain region. However, 13 of the 15 who initially respondedexhibited a recurrence of their symptomatology. With recurrentsymptomatology repeat rTCMS was effective again in improving symptomsbut only after application at the same site at which initial improvementoccurred, mainly the left temporoparietal region, a locus contralateralto patient handedness. This result indicates enhanced cognitiveprocessing following rTCMS in one brain locus, mainly left prefrontalcortex. Repeated stimulation in patients at this effective locusprolonged improvement in cognitive function. TCMS can be applied in manydifferent paradigms including use of single or repeated pulses, short orprolonged applications, varied wave forms, application intensity and amultiplicity of other parameters. The results of this experimentindicate that rTCMS improved sensory acuity and decreased sensorydistortions in patients with these cognitive impairments.

TABLE 42 Changes in taste and smell acuity in 17 patients withhypogeusia, hyposmia, phantosmia and/or phantageusia pre and post rTCMSI compared to normal responses TASTANT NaCl SUCROSE HCl UREA DT RT ME DTRT ME DT RT ME DT RT ME PRE    5.7 ± 0.7^(†a) 6.3 ± 0.9^(b )    26 ±18^(d‡)  4.9 ± 0.6^(c)  5.2 ± 0.6^(a) 29 ± 12^(c) 5.3 ± 0.9 6.8 ±0.8^(a) 27 ± 6^(a)  6.8 ± 1.4^(e) 9.0 ± 1.0^(a)  30 ± 9^(b) POST  3.2 ±0.0^(g) 3.6 ± 0.3^(b)* 35 ± 22 3.6 ± 0.4 3.9 ± 0.3 46 ± 12  4.1 ± 0.74.3 ± 0.6^(j) 57 ± 7^(k) 5.0 ± 1.0 5.7 ± 0.6^(h) 53 ± 8 NORMALS 3.3 ±0.3 3.4 ± 0.2  68 ± 4  3.3 ± 0.2 3.4 ± 0.2 60 ± 4   3.4 ± 0.4 3.5 ± 0.4 66 ± 4  3.6 ± 0.4 3.7 ± 0.4  68 ± 4 ODORANT PYRIDINE NITROBENZENETHIOPHENE AMYL ACETATE DT RT ME DT RT ME DT RT ME DT RT ME PRE   4.0 ±0.9^(†) 8.5 ± 0.7^(c)    35 ± 13^(e‡) 6.4 ± 0.7^(b) 9.4 ± 0.4^(a)  21 ±7^(b) 3.8 ± 0.8  7.4 ± 1.0^(a) 30 ± 7^(b) 4.3 ± 1.1 8.9 ± 1.8^(a) 24 ±7^(a) POST  1.9 ± 0.5^(ah) 4.4 ± 0.9^(f) 67 ± 11 3.2 ± 0.8^(g) 6.2 ±1.0^(g) 42 ± 8  1.9 ± 0.3^(i) 5.1 ± 0.9 45 ± 7^(h)   1.4 ± 0.0^(ch) 5.2± 0.9^(g) 44 ± 6^(f) NORMALS 3.7 ± 0.3 6.0 ± 0.7  66 ± 5  3.6 ± 0.4  6.0± 0.6  52 ± 6 3.2 ± 0.6 3.3 ± 0.5 69 ± 6  3.1 ± 0.5 3.3 ± 0.6  53 ± 5 DT, detection threshold (in BU), RT, recognition threshold (in BU), ME,magnitude estimation (in %) ^(†)MEAN ± SEM (in BU) ^(‡)MEAN ± SEM (in %)*All significance determined by Student's t test Normals are 150 normalvolunteer Compared to normals ^(a)p < 0.001 ^(b)p < 0.005 ^(b)*p < 0.01^(c)p < 0.02 ^(d)p < 0.025 ^(e)p < 0.05 Compared to pre rTCMS I ^(f)p <0.001 ^(g)p < 0.005 ^(h)p < 0.01 ^(i)p < 0.025 ^(j)p < 0.02 ^(k)p < 0.05

TABLE 43 Changes in phantageusia and phantosmia in 17 patients withhyposmia, hypogeusia, phantosmia and/or phantageusia pre and post rTCMSI PHANTAGEUSIA PHANTOSMIA PATIENTS PRE POST PRE POST TOTAL (17) 82 ±7^(†) 21 ± 7^(a) 72 ± 14 22 ± 12^(c) MEN (5) 71 ± 15 20 ± 15^(d) 70 ± 200^(b) WOMEN (12) 85 ± 6 22 ± 9^(a) 74 ± 18 33 ± 17  ( ) Patient Number^(†)MEAN ± SEM [in %, of most intense distortions experienced throughoutwaking state] $ Significance determined by Student's t test * Allresults compared to pre rTCMS I ^(a)p < 0.001 * ^(c)p < 0.02 * ^(d)p <0.025 * ^(e)p < 0.05 * ^(b)p < 0.01

TABLE 44 Changes in taste and smell acuity in 13 patients with hyposmia,hypogeusia, phantosmia, and/or phantageusia pre and post second rTCMStreatment TASTANT NaCl(4) SUCROSE(5) HCl(8) UREA (12) DT RT ME DT RT MEDT RT ME DT RT ME PRE   5.3 ± 1.5^(†)* 9.5 ± 2.1^(a) 50 ± 10^(‡) 4.8 ±0.7^(d) 7.3 ± 1.6^(c)* 46 ± 8 4.9 ± 0.5^(c)* 8.8 ± 0.8^(a ) 46 ± 8^(c)6.4 ± 0.3^(a) 7.9 ± 0.3^(a )   42 ± 10^(c)* POST 1.0 ± 0^(ae ) 6.2 ±2.8  68 ± 9   3.2 ± 0.7  4.7 ± 1.0   62 ± 8 4.1 ± 0.7  5.9 ± 0.9^(ch) 72± 6^(g) 3.4 ± 0.5^(e) 4.2 ± 0.9^(hm) 63 ± 11 NORMALS 3.3 ± 0.3 3.4 ±0.2  68 ± 4   3.3 ± 0.2  3.4 ± 0.2   60 ± 4 3.4 ± 0.4  3.5 ± 0.4  66 ±4  3.6 ± 0.4  3.7 ± 0.4   68 ± 4  ODORANT PYRIDINE(9) NITROBENZENE(10)THIOPHENE(10) AMYL ACETATE(12) DT RT ME DT RT ME DT RT ME DT RT ME PRE   6.2 ± 1.2^(i†) 10.0 ± 1.1^(b)    48 ± 10^(‡) 8.0 ± 1.9^(c) 10.3 ±0.6^(e)  39 ± 8 9.0 ± 1.8^(a) 10.0 ± 0.4^(a  )  57 ± 9 8.7 ± 0.4^(a)10.7 ± 0.4^(a)  36 ± 10 POST 4.8 ± 1.1 5.5 ± 1.6^(i) 76 ± 5^(g) 4.2 ±1.6  4.0 ± 14^(b)  62 ± 8 3.0 ± 0.9^(e) 5.0 ± 1.4^(bm) 70 ± 6 4.0 ±2.0^(I) 4.6 ± 1.5^(e) 51 ± 10 NORMALS 3.7 ± 0.3 6.0 ± 0.7  66 ± 5  3.6 ±0.4  6.0 ± 0.6  52 ± 6 3.2 ± 0.6  3.3 ± 0.5   69 ± 6 3.1 ± 0.5  3.3 ±0.6  53 ± 5  ^(†)MEAN ± SEM (in BU) ^(‡)MEAN ± SEM (in %) *Allsignificance determined by Student's t test Compared to normals ^(a)p <0.001 ^(b)p < 0.005 ^(c)p < 0.025 ^(l)p < 0.02 = c* ^(d)p < 0.05 ^(e)p <0.001 Compared to pre rTCMS II ^(y)p < 0.01 ¢ = m* ^(f)p < 0.05 ^(g)p <10.02 ^(h)p < 0.025 ^(I)p < 0.05 ^(m): < 0.005 m

TABLE 45 Changes in phantageusia and phantosmia in 13 patients withhyposmia, hypogeusia, phantosmia and/or phantageusia pre and post rTCMSII PHANTAGEUSIA PHANTOSMIA PATIENTS PRE POST PRE POST TOTAL (13) 39 ±10^(†l) 9 ± 5^(g) 47 ± 8 0^(a) MEN (4) 52 ± 15 6 ± 5^(h)  49 ± 10 0^(f)WOMEN (9) 34 ± 6^(k) 12 ± 6^(g)  44 ± 5 0^(f) ^(†)MEAN ± SEM [in %, ofmost intense distortions experienced throughout waking state] $Significance determined by Student's t test * All results compared topre rTCMS II and pre and post rTCMS I ^(a)p < 0.001 with respect to prerTCMS II and post rTCMS I ^(f)p < 0.001 compared to pre rTCMS II andpost rTCMS I ^(k)p < 0.001 compared to pre rTCMS I ^(l)p < 0.005compared to pre rTCMS I ^(g)p < 0.02 compared to pre rTCMS II ^(h)p <0.025 compared to pre rTCMS II

What is claimed is:
 1. A method of treating chemosensory dysfunction ina subject, the method comprising nasal administration of apharmaceutical composition comprising an effective amount of aphosphodiesterase inhibitor to the subject.
 2. The method of claim 1,wherein the phosphodiesterase inhibitor is theophylline.
 3. The methodof claim 1, wherein the phosphodiesterase inhibitor is cilostazol. 4.The method of claim 1, wherein the pharmaceutical composition comprisesa pharmaceutically acceptable aqueous solvent.
 5. The method of claim 1,wherein the pharmaceutical composition is a solution or suspension. 6.The method of claim 1, wherein the pharmaceutical composition is apowder.
 7. The method of claim 1, wherein the chemosensory dysfunctionis ageusia, hypogeusia, dysgeusia, anosmia, hyposmia, dysosmia, or acombination thereof.
 8. The method of claim 1, wherein the chemosensorydysfunction is ageusia, hypogeusia, dysgeusia, or a combination thereof.9. The method of claim 1, wherein the chemosensory dysfunction isanosmia, hyposmia, dysosmia, or a combination thereof.
 10. The method ofclaim 1, wherein the chemosensory dysfunction is smell loss, taste loss,or a combination thereof.
 11. The method of claim 1, wherein thechemosensory dysfunction is taste loss.
 12. The method of claim 1,wherein the chemosensory dysfunction is smell loss.
 13. The method ofclaim 1, wherein the subject has a decreased level of a cyclicnucleotide in a nasal mucus sample from the subject in comparison to acyclic nucleotide level of a control population with normal chemosensoryfunction.
 14. The method of claim 13, wherein the cyclic nucleotide iscAMP.
 15. The method of claim 13, wherein the cyclic nucleotide is cGMP.16. The method of claim 2, wherein the chemosensory dysfunction is tasteloss.
 17. The method of claim 3, wherein the chemosensory dysfunction istaste loss.
 18. The method of claim 2, wherein the chemosensorydysfunction is smell loss.
 19. The method of claim 3, wherein thechemosensory dysfunction is smell loss.